Method and system for sample preparation

ABSTRACT

A method for preparing a sample by utilizing a shearing force in the presence of a size stabilizer to break apart the sample to obtain nucleic acid molecules in a usable size range. Once nucleic acid molecules are obtained, magnetic nanoparticles are used to concentrate and clean the nucleic acid molecules for further testing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional patentapplication Ser. No. 14/056,603, filed Oct. 17, 2013, now U.S. Pat. No.9,347,086, which claims priority from U.S. Provisional PatentApplication Ser. No. 61/715,003 (filed Oct. 17, 2012), which is acontinuation-in-part of U.S. patent application Ser. No. 12/785,864,filed May 24, 2010, now U.S. Pat. No. 8,663,918, which claims priorityfrom U.S. Provisional Patent Application Ser. No. 61/180,494, filed May22, 2009, and which is also a continuation-in-part of U.S. patentapplication Ser. No. 12/754,205, filed Apr. 5, 2010, now U.S. Pat. No.8,716,006, which claims priority from U.S. Provisional PatentApplication Ser. No. 61/166,519, filed Apr. 3, 2009. The contents of theaforementioned applications are hereby incorporated by reference intheir entirety.

GOVERNMENT LICENSE RIGHTS

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of one or moreof the following Grant Award Nos. DMI-0450472 and IIP-0450472 awarded byNational Science Foundation, Contract No. W81XWH-07-2-0109 awarded by USArmy Medical Research and Material Command, Contract Nos.W911NF-06-1-0238 and W911NF-09-C-0001 awarded by US Army RDECOM ACQ CTR.

FIELD OF THE INVENTION

This invention relates to a method and system for analyzing biologicalsamples. More particularly, this invention relates to multi-chambervalves, and more particularly to multi-chamber disposable cartridges foruse in biological sample analysis.

BACKGROUND OF THE INVENTION

There is continuing interest to improve testing methodologies anddecrease time demands on clinical laboratories. Particular testingrequires that a sample be disrupted to extract nucleic acid moleculessuch as DNA or RNA.

It is estimated that about 30 million molecular diagnostic tests tookplace in US medical facilities in 2007. This figure is expected toincrease to 67 million in 2009. Many, if not all of these assays, couldbenefit from a rapid sample preparation process that is easy to use,requires no operator intervention, is cost effective and is sensitive tosmall size samples.

The use of molecular diagnostics and gene sequencing in research andmedical diagnostics are rapidly growing. Molecular techniques providehigher levels of specificity and sensitivity than antibody methods.Genetic sequencing allows for the collection of large amounts ofinformation not previously available. However, sample preparation is amajor cost component of running PCR (polymerase chain reaction),real-time PCR, gene sequencing analysis and hybridization testing. Inaddition, it delays test results and limits the ability to run theseassays to laboratories with well trained personnel.

Nucleic acid based identification of biological material first requiresisolation of the nucleic acid molecules (NAMs) from the sample. In orderfor a system to effectively and efficiently meet the user's needs, auniversal sample preparation process is required. Current samplepreparation processes are laborious, time consuming and requirelaboratory capability.

Therefore, there is a need for an improved testing system andmethodology that addresses at least some of these shortcomings.

SUMMARY OF THE INVENTION

The present invention relates to a sample preparation device. The samplepreparation module is designed to identify and validate components forultrasonic disruption and magnetic manipulation of nucleic acidmolecules. In one embodiment, all processing steps occur within adisposable cartridge.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is disclosed with reference to the accompanyingdrawings, wherein:

FIGS. 1A-1B show a graphical representation of a disposable cartridgeaccording to one embodiment;

FIG. 2 shows an expanded view of a disposable cartridge according to oneembodiment;

FIG. 3A shows a cross-sectional view of a disposable cartridge accordingto one embodiment;

FIG. 3B shows a cross-sectional view of a disposable cartridge accordingto one embodiment having a magnet and sonicator in the cartridge;

FIGS. 4A-4D show a graphical representation of the cartridge bodyaccording to one embodiment;

FIGS. 5A-5B show a cross-sectional view of an assembled disposablecartridge according to one embodiment having the multi-chamber insertsecured in the cartridge body.

FIGS. 6A-6G show a graphical representation of the multi-chamber insertaccording to one embodiment;

FIGS. 7A, 7B, 8A, 8B, 9A, 9B, 10A, 10B, 11A, 11B, 12A, 12B, 12C, 13A,13B, 14A, 14B, 14C, 15A, 15B, 16A, 16B and 16C show various graphicalrepresentations of an assembled disposable cartridge with themulti-chamber insert positioned for desired fluid flow through thechannels and ports according to one embodiment;

FIG. 17 shows a schematic representation of a disposable cartridgeaccording to one embodiment;

FIG. 18 shows a process flow chart for one use of a disposable cartridgeaccording to one embodiment;

FIGS. 19A, 19B, 20A, 20B, 21A and 21B show a graphical representation ofmulti-chamber insert configurations according to various embodiments;

FIG. 22 shows a graphical representation of sampling device containing acartridge drive and plunger drive according to one embodiment;

FIG. 23 shows a graphical representation of a cartridge drive with thedisposable cartridge removed according to one embodiment;

FIG. 24 shows a graphical representation of the stepper motor assemblyand worm drive according to one embodiment;

FIG. 25 shows a graphical representation of a heater according to oneembodiment;

FIG. 26 shows a graphical representation of a disposable cartridgeaccording to one embodiment;

FIG. 27 is a top perspective view of an exemplary disposable cartridge;

FIG. 28A is a bottom view of the exemplary disposable cartridge of FIG.27;

FIG. 28B, FIG. 29A and FIG. 29B are depictions of components that alignwith the bottom of the exemplary disposable cartridge;

FIG. 30 is an alternate top perspective view of an exemplary disposablecartridge;

FIG. 31A, FIG. 31B and FIG. 31C are exemplary systems for preparing anucleic acid sample;

FIG. 32 is an exemplary systems for preparing a nucleic acid sample;

FIG. 33 demonstrates the effective release of nucleic acid moleculesfrom the lysis of spores using ultrasonic bead beating with sizestabilizer;

FIG. 34 demonstrates nucleic acid molecules isolated from fruit fliesand that the addition of a size stabilizer in lanes two and threeprotect the nucleic acid molecules from over-shearing, whereas thesamples without the denaturants were sheared to a level well below 100base pairs;

FIG. 35 shows that using this process the nucleic acid molecules from awide variety of different samples can be treated with the same powerlevels and time of sonication to give the same size distribution offragments;

FIG. 36 is a graphical representation showing the release of the nucleicacid molecules from the magnetic nanoparticles;

FIG. 37 demonstrates the nucleic acid molecule isolation obtained fromusing tissue from the ear of a cow;

FIG. 38 demonstrates the nucleic acid molecule isolation obtained fromusing fruit flies contaminated with soil;

FIG. 39 demonstrates purified DNA recovered from fruit flies;

FIG. 40 demonstrates DNA recovered from fruit flies using variousbuffers;

FIG. 41 demonstrates the recovery of nucleic acid molecules from yeast,grass and blueberries;

FIG. 42 demonstrates the recovery of nucleic acid molecules from e. colishowing longer sonication times do not change the size distribution;

FIG. 43 is a graphical representation of DNA recovery from increasingvolumes of a bacterial cell culture using the instant invention, thecommercial QIAGEN kit for DNA recovery and the textbookPhenol/Chloroform method;

FIG. 44 demonstrates the effectiveness of high ionic strength buffer inprotecting nucleic acid molecules during sonication;

FIG. 45 demonstrates that sonication in the presence of a selected sizestabilizer can provide a high yield of DNA in a limited size range;

FIG. 46 depicts an exemplary cartridge;

FIG. 47A is a cutaway view of an exemplary drive assembly; FIG. 47B is abottom view of an insert while FIG. 47C is a cut away view of theinsert;

FIG. 48A and FIG. 48B are views of a first exemplary insert pod whileFIG. 48C and FIG. 48D are views of a second exemplary insert pod;

FIG. 49 is a perspective view of a drive platform;

FIG. 50 is a depiction one an exemplary cover for sample pre-processing;

FIG. 51 shows a device for liquid sample collection;

FIG. 52 illustrates an exemplary multi-sample collection disk;

FIG. 53 depicts a cover that uses an absorbing solid to collect a liquidsample;

FIG. 54 depicts an alternate embodiment of a cover that uses anabsorbent solid;

FIG. 55A, FIG. 55B and FIG. 55C are depictions of a lance-based systemfor collecting a liquid sample; and

FIG. 56 is another embodiment of a lance-based system.

Corresponding reference characters indicate corresponding partsthroughout the several views. The examples set out herein illustrateseveral embodiments of the invention but should not be construed aslimiting the scope of the invention in any manner.

DETAILED DESCRIPTION

Referring to FIG. 1A and FIG. 1B there is shown an exemplary assembleddisposable cartridge 100. The disposable cartridge 100 comprises acylindrical insert 101. The cylindrical insert 101 is rotatably situatedwithin a cartridge body 102. The cylindrical insert 101 compriseschambers 103 for containing or treating fluid; a plurality of fluidpaths for connecting the chambers 103 to external ports; and fluidthrough channels for transmitting fluids.

The disposable cartridge 100 provides an automated process for preparinga biological sample for analysis. The sample preparation process of theinstant invention can prepare fragments of DNA and RNA in a size rangeof between 100 and 10,000 base pairs. The exact distribution of sizescan be varied by changing concentrations of surfactants, the surfactantsused or the frequency of sonication. The ability to produce fragments inthe desired size range obviates the need for electrophoresis or columnisolation. This also increases the overall yield of useful fragments byeliminating the need for addition purification steps. A samplepreparation module allows for disruption of cells, sizing of DNA andRNA, concentration and cleaning of the material. Additional chambers inthe cylindrical insert can be used to deliver the reagents necessary forend-repair and kinase treatment. Enzymes can be stored dry andrehydrated in the cartridge or added to the cartridge just prior to use.

The use of a rotating design allows for a single plunger to draw andpush fluid samples without the need for a complex valve system to openand close at various times. This greatly reduces potential for leaks andfailure of the device compared to conventional systems. Furthermore, theuse of a plunger allows for greater configurability in adjusting theamount of fluid drawn. The disposable cartridge 100 can be stored in arotary position that leaves all ports and vents closed. This allows forlong-term storage and shipping of the disposable cartridge 100 withliquid and solid reagents loaded within the disposable cartridge 100. Inuse, the disposable cartridge 100 is inserted into a detection devicethat is in electrical communication with a chip 107 (see FIG. 2). Thedetection device further affixes the cartridge body 102 into a fixedposition.

Referring to FIG. 2 there is shown an exploded view of the disposablecartridge 100. The cylindrical insert 101 is capable of containing aplurality of fluids in the various chambers 103. The exterior of thecylindrical insert 101 is cylindrical to allow free rotation about itsaxis when encased in the cartridge body 102. The interior section of thecylindrical insert 101 can be modified to include any size or shapechamber. Customized disposable cartridges retain the same exterior shapeand dimensions and can be inserted into existing detection devices. Theprocessing protocol of the detection device is easily modified toaccount for any new chambers, sample sizes, processing times, or portlocations. In one embodiment, the cylindrical insert 101 is formed by aninjection molding technique. In another embodiment, both the cylindricalinsert 101 and the cartridge body 102 are formed through injectionmolding techniques. Injection molding allows for the production ofcustomized disposable cartridges with minimal costs. The disposablecartridge 100 is configured to allow fluid contained in the chambers 103to pass through certain fluid paths. The design allows for easymanufacturing and assembly. The design further allows for the disposablecartridge 100 to be used in instruments requiring a plurality of fluids.In one embodiment, the disposable cartridge 100 is a single use piecefor use in detection devices. The disposable cartridge 100 contains thenecessary fluids for biological testing and further is capable of beinginjected with a field sample.

Referring again to FIG. 2, the heat seal films 104 seal the fluids intothe cylindrical insert 101 and prevent leaks while allowing for themanipulation of fluid samples. The heat seal films 104 seal the chambers103 from the outside environment. The heat seal films 104 further allowfor fluid to be added to or removed from the chambers 103 withoutcompromising the integrity of the seal. In one embodiment, the heat sealfilms 104 improve energy transfer into and out of the chambers 103 ofthe cylindrical insert 101. Energy transfer includes but is not limitedto heat, ultrasonic and magnetic. In one embodiment, a filter 105 isplaced in-line with particular fluid paths to filter large solids fromthe fluid. In one embodiment, once the heat seal films 104 are sealedonto the cylindrical insert 101, the cylindrical insert 101 is affixedto the cartridge body 102. In one embodiment, the cylindrical insert 101snaps into the cartridge body 102. It is understood that the heat sealfilms 104 can be sealed to the cylindrical insert 101 after thecylindrical insert 101 is affixed to the cartridge body 102.

In one embodiment a chip 107 containing biological probes is affixed tothe cartridge body 102. The fluid contained in the chambers 103 istransferred to contact the chip 107 containing biological probesinitiating reaction or detection chemistry. The chip 107 is incommunication with a detection device, such as a bench-top detectiondevice or portable detection device, to indicate the presence of atarget analyte in a sample.

Referring now to FIG. 3A and FIG. 3B there is shown a cross sectionalview of the disposable cartridge 100. The disposable cartridge 100 isset onto a cartridge drive 110. The cartridge drive 110 is capable ofrotating the cylindrical insert 101 to a desired rotary position. Thecartridge drive 110 rotates the cylindrical insert 101 while thecartridge body 102 remains stationary. In one embodiment the cartridgedrive 110 has one or more heaters 111. The heater 111 is capable ofheating the fluids contained in the chambers 103 to a desiredtemperature. Alternatively, heating chambers are strategicallypositioned above the heater 111 to heat the fluid in the heating chamberwithout significantly heating the fluids in the chambers 103. In oneembodiment, the heat film seals 104 facilitate this heating withoutsignificantly heating the fluids in the chambers 103. Various treatmentchambers are incorporated into the cylindrical insert 101 to facilitatemixing, heating, disrupting, pressurization or any other treatmentprocess. In one embodiment, cartridge drive 110 includes a magnetic 114.The magnet 114 is utilized to generate a magnetic field. The magnet 114can pull or push magnetic nanoparticles in the cylindrical insert 101.The magnet 114 can concentrate a sample of magnetic nanoparticles orspeed up the diffusion process by guiding any magnetic nanoparticles.See the section of this specification entitled “magnetic manipulation.”

A mechanical force, such as a shearing force, is applied to a biologicalsample to disrupt the sample and cause it to release nucleic acidmolecules. In one embodiment, the sample material is shredded with highspeed nanoparticles utilizing sonication. This process disrupts cells,tissue or other materials to release nucleic acid molecules. It isunderstood that the mechanical force can be any force suitable fortearing apart the sample to release the nucleic acid molecules. Suitablemechanical forces include, but are not limited to sonication,nebulization, homogenization, etc. Bead beating is a process to isolatenucleic acid molecules from samples. It is a robust approach which iswell suited for use with spores or tissue samples. In bead beating,glass beads of about 100 microns in diameter are used to crush thesample to release the nucleic acid molecules. The beads are moved usingan ultrasonic source. FIG. 33 demonstrates the effective release ofnucleic acid molecules from spore samples. In another embodiment,sharpened shards are used in place of, or in addition to, beads. Theseshards may be useful in releasing the nucleic acids from whole organisms(e.g. insect bodies) or similarly resilient structures.

For example, in one embodiment the cartridge drive 110 has a disruptor112. The disruptor 112 is capable of mixing or disrupting the fluidscontained in the chambers 103 by applying an ultrasonic force. Theexemplary disposable cartridge 100 has a disrupting chamber 113 formixing fluids in a chamber distinct from the chambers 103. In oneembodiment small beads are located in the disrupting chamber 113 or inone of the chambers 103 to assist in mixing fluids or breaking downsamples. The disrupter 112 applies an ultrasonic force causing the beadsto become excited and move through the fluid.

A size stabilizer is present during the disruption step to obtainnucleic acid molecules within a usable size range. In one embodiment,the nucleic acid molecules are reduced to sizes between 200 and 10,000base pairs in length. In another embodiment the nucleic acid moleculesare reduced to sizes between 300 and 3,000 base pair in length. Inanother embodiment the nucleic acid molecules are reduced to sizesbetween 400 and 2,000 base pair in length. In another embodiment thenucleic acid molecules are reduced to sizes between 200 and 500 basepair in length. It is understood that the desired base pair length willvary depending on the downstream sample processing technique. Sampleprocessing techniques include, but are not limited to hybridization,PCR, real-time PCR, reverse transcription-PCR, “lab-on-a-chip” platformsand DNA sequencing.

Referring to FIG. 4A to FIG. 4D there are shown various views of oneembodiment of the cartridge body 102. It is understood that variousdesigns can be used to house the cylindrical insert 101. The cartridgebody 102 has an inner cylindrical surface 140. The inner cylindricalsurface 140 houses the cylindrical insert 101 (see FIG. 2). The innercylindrical surface 140 is smooth to allow the cylindrical insert 101 tofreely rotate. The cartridge body 102 is constructed from any materialthat is both ridged enough to support the cartridge body 102 and smoothenough to allow for rotation of the cylindrical insert 101. In oneembodiment, the inner cylindrical surface 140 has a slight taper tofacilitate attachment of the cylindrical insert 101 that also has anouter cylindrical surface with a slight taper.

As shown in FIG. 4A to FIG. 4D, in one embodiment the cartridge body 102has a syringe molding 141. Although only one syringe molding is shown itis understood that a plurality of syringe moldings can be used. In theembodiment of FIG. 4C, the syringe molding 141 is a hollow pipe thatextends perpendicular from the vertical edge of cartridge body 102. Thesyringe molding 141 is capable of housing a plunger. The plunger drawsand pushes fluids through the fluid paths of cylindrical insert 101.

Referring to FIG. 5A and FIG. 5B there is shown a cross sectional viewof the assembled disposable cartridge 100 having a plunger 150. Theplunger 150 is capable of drawing fluid from the chambers 103. Once theplunger 150 draws the fluid, the disposable cartridge repositions thefluid path to align a distinct port with the syringe molding 141 whichis in fluid communication with a reaction chamber 142 or a differentchamber 103. The plunger 150 then pushes the fluid through the fluidpath 151 into the reaction chamber 142 or the different chamber 103. Inone embodiment the plunger 150 is retained within the syringe molding141. The fluids chemically react with other fluids or devices incommunication with the reaction chamber 142 where it contacts the chip107 (see FIG. 2). In one embodiment the chip 107 has a reactive surfaceand is mounted on a sensor board. In one embodiment the chip 107 formsone side of the reaction chamber 142. The chip 107 is in electricalcommunication with a detection device to provide readings and results ofthe testing. As shown in FIG. 4D, a sensor mount 143 is capable ofholding the sensor board. The sensor board is aligned to the sensormount 143 by the alignment posts 146.

It is understood that a fluid output can be attached to the cartridgebody 102 to allow the fluid to transfer from the disposable cartridge100 to a desired location. Furthermore, a fluid input allows theintroduction of fluids to the disposable cartridge 100. While a plunger150 has been described in this embodiment, it is understood that anysuitable fluid delivery device could be substituted to effectivelytransfer fluids within the cartridge.

Referring to FIG. 6A to FIG. 6G there are shown multiple views of thecylindrical insert 101. The chambers 103 of cylindrical insert 101 cancontain samples, standards, washes, catalysts or any other desirablefluids. In one embodiment the chambers 103 include a waste chamber tohold discharged fluids. The cylindrical insert 101 further containsmultiple ports 160. Each port 160 has a unique fluid path. Each chamberhas a fluid path that is in communication with a port to transfer fluidto or from the chamber. A syringe molding on the cartridge body (notshown) aligns with a port to extract or push fluid. To prevent pressuredifferentials from forming, pressure relief ports 164 are positionedalong the cylindrical insert. In addition to the unique fluid paths, thecylindrical insert 101 contains at least one fluid through-channel 161.The fluid through channel 161 is an elongated channel that traverses atleast a portion of the bottom edge of the cylindrical insert 101 andallows the fluid to flow from the one end of the cylindrical insert 101to the other. For example, the fluid can flow from the syringe molding141, through a fluid through channel, and into the reaction chamber 142of the cartridge body 102. To prevent fluid interaction in the fluidthrough channel 161 a plurality of fluid through-channels are used. Asecondary fluid through-channel 162 is used to prevent early reactionsor other adverse fluid interactions. In one embodiment the cylindricalinsert 101 contains a heater contact region 163. The heater contactregion 163 is positioned below the chambers 103 for which it isdesirable to heat the fluid in the chamber. Furthermore, the heater 111(see FIG. 3A) is capable of heating the fluid through channel 161.

Referring to FIG. 7A to FIG. 16C there are shown multiple of views of anassembled disposable cartridge rotated in various positions. As shown inFIG. 7A and FIG. 7B the cylindrical insert 101 is in a closed position.No ports 160 are aligned with the syringe molding 141. This prevents anyleakage of fluid from the chambers 103. In one embodiment at least onechamber 103 is a sample chamber. The sample chamber enables the user toinject a fluid sample into the chamber through the heat film seal. Inone embodiment the sample chamber contains disrupting objects, such asglass beads, to assist in breaking down samples into testable nucleicacid strands.

Referring to FIG. 8A and FIG. 8B the cylindrical insert 101 has a rotaryposition such that port 3P is in-line with the syringe molding 141. Oncepositioned fluid from a chamber 3R that is fluidly connected to port 3Pcan be drawn through port 3P and into the syringe molding 141. Oncefluid is pulled from the chamber 3P, and no additional fluid is requiredfrom that chamber, that chamber can be used as an alternative chamberfor waste storage.

Referring to FIG. 9A and FIG. 9B, the cylindrical insert 101 has arotary position such that port 11P is aligned with the syringe molding141. In the embodiment depicted, port 11P is fluidly connected toreaction chamber 142. The plunger 150 pushes the fluid within thesyringe molding 141 into port 11P and the fluid passes to the reactionchamber 142.

Referring to FIG. 10A and FIG. 10B the cylindrical insert 101 ispositioned such that port 8P is aligned with the syringe molding 141. Inone embodiment fluid is pushed from the syringe molding 141 into port 8Pand into a chamber 103 disposed proximate a heating chamber 170. Once inthe heating chamber 170 the fluid is heated at the desired temperaturefor a predetermined amount of time.

Referring to FIG. 11A and FIG. 11B, once the heating is completed thefluid is drawn back into the syringe molding 141. It is understood thatthe fluid may be drawn through the same port 8P or unique port incommunication with the heating chamber 170. As shown in FIG. 11A andFIG. 11B the fluid is drawn into the syringe molding 141 from a uniqueport 9P in communication with the heating chamber 170.

Referring now to FIG. 12A to FIG. 12C there is shown a fluid path fromthe syringe molding 141 to the reaction chamber 142. In this embodimentthe reaction chamber 142 is fluidly connected with port 11P.

Referring to FIG. 13A and FIG. 13B there is shown the cylindrical insert101 positioned such that port 14P is aligned with the syringe molding141. Chamber 14R is in communication with port 14P. The fluid containedin chamber 14R is pulled into the syringe molding. The cylindricalinsert 101 then rotates to port 13P as shown in FIG. 14A and FIG. 14C.The fluid from chamber 14R is then pushed through port 13P to thereaction chamber 142. The fluid passes through a channel that isdistinct from the channel associated with port 11P. This prevents fluidsfrom coming in contact with and reacting with each other while in thechannels. The fluids first come into contact in the reaction chamber142.

After the desired reaction time the plunger 150 draws the fluid from thereaction chamber 142 and pushes the fluid into the waste chamber 7. Theplunger 150 draws the fluid back through port 11P and the cylindricalinsert 101 rotates to a port in communication with waste chamber 7. Theplunger 150 then pushes the fluid into the waste chamber 7. It isunderstood that after use any chamber can be utilized as a wastechamber. In an alternative embodiment, the plunger 150 stops pushingfluid once it reaches the reaction chamber 142. Upon completion of thereaction time, the plunger 150 continues to push the fluid through thereaction chamber 142 and into a port in communication with a wastechamber or separate archive chamber. An archive chamber stores thesample for additional testing or verification.

Referring to FIG. 15A and FIG. 15B there is shown the cylindrical insert101 positioned such that port 4P is aligned with the syringe molding141. Port 4P is in communication with chamber 4R containing a flushingfluid. The flushing fluid is drawn from chamber 4R through port 4P andinto the syringe molding.

As shown in FIG. 16A, FIGS. 16B and 16C, the cylindrical insert 101rotates to port 11P and the plunger pushes the flushing fluid into port11P and to the reaction chamber 142.

Once processing is completed the disposable cartridge 100 can be removedfrom the detection device and disposed. A fresh disposable cartridgewith the same or different configuration is then inserted into thedetection device in preparation for the next use.

Referring to FIG. 17 there is shown a schematic of a disposablecartridge of one embodiment. The exemplary cylindrical insert containssix fluids in various chambers. Five fluids pass from their respectivechambers, into the syringe molding, through the main channel 180 andinto a reaction chamber, such as reaction chamber 142. One fluid passesfrom the syringe molding through a secondary channel 181 and into thereaction chamber to prevent any contamination or premature reactions.

Referring to FIG. 18 there is shown a process flow according to oneembodiment. Once a sample is injected into a sample chamber thedetection device is activated and the testing begins. The channels arefirst preconditioned with a small amount of buffer. The sample is thentransferred from the sample chamber to a heating chamber and heated at95° C. for 5 minutes. The heated sample is then transferred to areaction chamber to hybridize for 20 minutes. The hybridization processenables the sample to chemically bond with biological probes found on achip in communication with the reaction chamber. The biological probesspecifically bind to target nucleic acid molecules found in the sampleas described in U.S. Pat. No. 6,399,303 issued to Connolly on Jun. 4,2002, which is hereby incorporated by reference. It is understood that asingle chip may contain a plurality of distinct and redundant biologicalprobes to increase sensitivity and to test for a variety of targetnucleic acid molecules. It is further understood that the disposablecartridge can be used in any system requiring the manipulation andtransport of a plurality of fluids.

After hybridization, the sample is flushed with buffer to remove anyexcess compounds. In one embodiment, a catalyst such as palladium istransferred to the reaction chamber and allowed to incubate for 10minutes. The remaining catalyst is then flushed with water. A mixture ofa reducing agent and metal, such as nickel, is pushed into the reactionchamber. The metal coats the target sample creating a conductor on thechip. The excess non-bonded metal is flushed with water. The resistanceacross biological probes bonded together by a target sample coated inmetal dramatically reduces, indicating the presence of the targetsample. The detection device writes the results of the test and the testis complete.

Referring now to FIG. 19A and FIG. 19B there are shown variations of thecylindrical insert. The chambers of the insert are shown in arectangular configuration. Changes to the chamber sizes and shapes canbe performed to optimize the particular reagent and waste chamber.

Referring now to FIG. 20A and FIG. 20B there are shown additionalvariations of the cylindrical insert. The chambers of this embodimentare shown to have radial chambers. In one embodiment the chambers are ofuniform size and shape around the radius of the insert.

Referring now to FIG. 21A and FIG. 21B there are shown variations of thecylindrical insert. The chambers are of various sizes along the radiusof the insert to house differing amounts of reagents within eachchamber. While variations of the insert are shown in the variousembodiments, it is understood that any variation of the cylindricalinsert containing a plurality of ports and chambers can be used.

Referring to FIG. 22 there is shown a sampling device having a plungerdrive 220 and a cartridge drive (also see FIG. 23). The plunger drive220 contains a long cylindrical section 221 having a tip 220. The tip ofthe plunger drive 220 connects to the plunger inside of the syringemolding 141. In one embodiment the tip of the plunger drive 220 isconical to improve contact with the plunger. The plunger drive 220 movesthe cylindrical section 221 axially causing the plunger to either pullor push fluids from the chambers in the disposable cartridge 100. Thedisposable cartridge 100 sets on top of the cartridge drive.

Referring to FIG. 23 there is shown a cartridge drive according to oneembodiment. The disposable cartridge 100 sets atop the contact surface230. The contact surface 230 rotates to position the cylindrical insert101 to a desired location within the disposable cartridge 100. In oneembodiment the contact surface 230 is part of a drive assembly 231. Aworm gear 232 is attached to the drive assembly 231. A worm drive 233engages the worm gear 232 causing the drive assembly 231 to rotate. Itis understood that any suitable means to rotate the cylindrical insert101 can be employed.

Referring to FIG. 24 there is shown another view of the cartridge drive.The worm drive 233 is a stepper motor positioned to advance the wormgear 232. A home flag 240 is attached to the drive assembly to zero thedevice. At any time during fluid sampling the home flag can be zeroedallowing the worm drive 233 to advance the appropriate distance.

Referring to FIG. 25 there is shown the contact surface 230 having aheater. The contact surface 230 is spring loaded to improve contact withthe disposable cartridge 100. At least one spring 254 is positioned toallow movement of the contact surface 230. In one embodiment the contactsurface 230 contains a heater mount 250 to mount the heating elements.At least one resistor 251 is positioned on the heater mount 250. Aheating plate 252 transfers heat from the resistor 251 through theheating plate 252 and to a desired location on the disposable cartridge100. In one embodiment the heating plate 252 is an aluminum heatingplate. In one embodiment, a temperature sensor 253 is positioned nearthe resistor 251 or heating plate 252 to detect the resultingtemperature. It is understood that the contact surface 230 can bepositioned over the heater plate. The contact surface is made from amaterial that allows an efficient thermal transfer from the heatingplate to the disposable cartridge.

Referring to FIG. 26, disposable cartridge 300 is depicted. Disposablecartridge 300 is similar to disposable cartridge 100 except in that adifferent cylindrical insert 302 is used. The cylindrical insert 302 isdisposed within an inner cylindrical surface of cartridge body 304 andis rotatably connected thereto. The cylindrical insert 302 comprises aplurality of ports 306, each of which is connected to a correspondingchamber. In the embodiment of FIG. 26, each of the ports 306 are at thesame predetermined height along the vertical edge of cylindrical insert302. This permits each of the ports 306 to be selectively aligned with asingle syringe mold 308. By rotating the cylindrical insert 302 relativeto the cartridge body 304, each individual port 306 can be selectivelyaligned with syringe mold 308, thereby permitting fluid to beselectively injected or withdrawn from a desired chamber. In FIG. 26,the ports 306 are on the vertical edge of the cylindrical insert 302. Inother embodiments, the ports 306 are disposed on other edges, such as atop or bottom edge.

Referring to FIG. 27, a top view of the cylindrical insert 302 is shown.Cylindrical insert 302 comprises a disrupting chamber 310 that isfluidly connected to a first port among the ports 306 (see FIG. 26). Thedisrupting chamber 310 of FIG. 27 is centered with respect tocylindrical insert 302. In other embodiments, the disrupting chamber 310may be disposed elsewhere in cylindrical insert 302. In the embodimentdepicted, the first port is connected to disrupting chamber 310 by afirst elongated channel that traverses a portion of the bottom edge ofthe cylindrical insert 302. The cylindrical insert 302 further comprisesat least one additional chamber. Examples of chambers include a wastechamber 312, a sample processing chamber 314 and a catalyst chamber 316.Additional chambers may hold buffer solutions, washing solutions,suspensions of magnetic nanoparticles, developer solutions, enzymaticsolutions including PCR reagents, dehydrated reagents and the like. Inone embodiment, one chamber is reserved for use as an archive chamberwherein processed nucleic acid molecules may be stored for an extendedperiod of time.

In the exemplary embodiment of FIG. 27, cylindrical insert 302 includesa column chamber 318. Column chamber 318 is formed by a first wall 320and a second wall 322. In the embodiment depicted, second wall 322 isshorter than first wall 320. The column chamber 318 is fluidly connectedto at least one port by an elongated channel that traverse at least aportion of the bottom edge of the cylindrical insert 302. The columnchamber 318 may be filled with a chromatography material, such as silicagel, that is suitable for column chromatography. Fluid may be pushedinto the lower portion of column chamber 318 through the elongatedchannel. The fluid passes through the chromatography material and beginsto fill column chamber 318. When the fluid reaches the high of secondwall 322, the chromatographed fluid flows into overflow chamber 324where it may be subsequently withdrawn via another port.

FIG. 28A is a bottom view of cylindrical insert 302. FIG. 28A showselongated channels that traverse at least a portion of the bottom edgeof the cylindrical insert 302. Elongated channel 400 fluidly connectsdisrupting chamber 310 to a port (not shown) in the edge of cylindricalinsert 302. Similarly, elongated channels 402, 404 and 406 also traverseat least a portion of the bottom edge. The elongated channels 402, 404and 406 have a volume which is sufficient to function as chambers butthe elongated channels 402, 404 and 406 extend parallel to the bottomsurface of the cylindrical insert 302 and are therefore proximate thecontact surface of the cartridge drive. Other elongated channels arealso shown in FIG. 28A.

FIG. 28B is a view of the contact surface 408 of the cartridge drive(not shown). The contact surface 408 and the cylindrical insert 302 havemated connectors 410 a/410 b which permit the contact surface 308 andthe cylindrical insert 302 to become fixedly connected, therebypermitting rotation of the cylindrical insert 302 when the contactsurface 408 is rotated. The contact surface 408 includes a disruptor412, such as an ultrasonic disruptor, which is aligned with thedisrupting chamber 310. Disposed beneath the rotatable contact surface408 is magnet 414, first heater 416 and second heater 418. The magnet414, first heater 416 and second heater 418 are fixedly mounted to thecartridge drive such that they do not rotate when contact surface 408 isrotated. Each is offset from the center of the cylindrical insert.Advantageously, this permits specific zones to be disposed near amagnetic field, a first heater or a second heater, simply by rotatingthe cylindrical insert 302.

By way of illustration, the cylindrical insert 302 of FIG. 28A has afirst zone 420, a second zone 422 and a third zone 424. The third zone424 may be exposed to the magnetic field of magnetic 414 by rotating thecylindrical insert 302 into the rotary position shown in FIG. 29A.Conversely, the third zone 424 may be removed from the magnetic field ofmagnet 414 by rotating the cylindrical insert 302 into the rotaryposition shown in FIG. 29B, which is a 180 degree rotation. In anotherembodiment, the third zone 424 may be removed from the magnetic field ofmagnet 414 with a 90 degree rotation to also place the third zone 424over one of the heaters, 416, 418.

In an analogous fashion, a sample may be introduced into elongatedchannel 404. Elongated channel 404 traverses both first zone 410 andsecond zone 422. The first zone 410 may be disposed over first heater416 (e.g. to achieve a temperature of 50-55° C.) while the second zone422 may be disposed over second heater 418 (e.g. to achieve atemperature of 90-95° C.) by adopting the rotary position shown in FIG.29A. The relative positioning of the zones may be reversed by adoptingthe rotary position shown in FIG. 29B. This configuration isparticularly advantageous when a PCR operation is conducted within oneor more of the elongated channels. The high and low temperature cyclingused in the PCR operation can be produced by rotating the cylindricalinsert 302 to place the elongated channel over high and low temperatureheaters. By repeatedly cycling the rotary positions, the sample withinthe elongated channels experiences multiple iterations of high and lowtemperatures.

In operation, and with reference to FIG. 30, a biological sample isdisposed in disrupting chamber 310 of cylindrical insert 302. Thecylindrical insert 302 is rotated to align port 504P with the plunger(not shown). The plunger is activated to withdraw a lysis buffersolution from chamber 504. The cylindrical insert 302 is then rotated toalign a port with the plunger that is in fluid communication with thedisrupting chamber 310. In the embodiment of FIG. 30, the fluidcommunication is established through elongated channel 400 (see FIG.28A). The plunger is activated to inject the lysis buffer into thedisrupting chamber 310. Ultrasonic force is applied from disruptor 112to disrupt the biological sample and release the nucleic acids. In oneembodiment, a size stabilizer is present to control the size of thefragments produced during the disruption step.

The cylindrical insert 302 is then rotated to align the plunger with theport that is in fluid communication with disrupting chamber 310. Theport may include an in-line filter, such as a 0.8 micron filter. Theplunger is activated to withdrawn the solution from disrupting chamber310 and simultaneously pass the solution through the filter.

The cylindrical insert 302 is then rotated to align port 314P with theplunger. The plunger is activated to inject the solution into processingchamber 314. Processing chamber 314 includes a suspension of magneticnanoparticles. The solution is exposed to the magnetic nanoparticles fora period of time that is sufficient to allow the nucleic acids to bindto the magnetic nanoparticles. The plunger is thereafter activated towithdraw the suspension of magnetic nanoparticles from processingchamber 314.

The cylindrical insert 302 is then rotated to align the plunger with aport that is in fluid communication with elongated chamber 402. Theplunger is activated to inject the suspension of magnetic nanoparticlesinto elongated chamber 402. The elongated chamber 402 traverses at leasta portion of a bottom edge of the cylindrical insert 302. The elongatedchamber 402 is disposed proximate to a magnet, such as magnet 414. Thismagnet causes the magnetic nanoparticles and the nucleic acids boundthereto, to become concentrated in a particular area within theelongated chamber 402. Advantageously, this holds the magneticnanoparticles in place while allowing unbound material to be rinsedaway. In one embodiment, elongated chamber 402 includes a wide region402 whose diameter is wider than the diameter of the other portions ofelongated chamber 402. When the magnetic field is applied, thenanoparticles concentrate in wide region 402 without clogging theelongated chamber 402, thereby permitting wash solutions to pass overthe concentrated nanoparticles.

Wash solutions for washing the magnetic nanoparticles may be withdrawnfrom other chambers. In one embodiment, the cylindrical insert 302 isrotated to align port 508P with the plunger. The plunger is activated towithdraw a wash solution from chamber 508. Examples of suitable washsolutions include water, ethanol, 70% ethanol, buffered solutions, andthe like. The cylindrical insert 302 is rotated to re-align the plungerwith the port that is connected to elongated chamber 402. The plunger isactivated to inject the wash solution into the elongated chamber 402. Asthe wash solution passes over the magnetic nanoparticles, excess liquidpasses through elongated chamber 402, out hole 312A and into chamber312. In the embodiment of FIG. 30, chamber 312 is a waste chamber. Thiswash step may be repeated as desired.

As a further advantage of the rotary approach, the rotation of thecylindrical insert 302 to withdraw the wash solution also moves theelongated chamber 402 away from the magnet. This permits the magneticnanoparticles to become resuspended which facilitates the removal ofunbound material that could have been caught between clumpingnanoparticles. When the cylindrical insert 302 is rotated into positionto inject the wash solution into the elongated chamber 402 then theelongated chamber 402 is once again proximate the magnet.

In one embodiment, the final wash is a release solution configured torelease the nucleic acids from the magnetic nanoparticles. After therelease solution has been allowed to contact the magnetic nanoparticlesfor a sufficient period of time, the plunger is activated to withdrawthe release solution and the dissolved nucleic acids. In one embodiment,the release solution is heated to promote release of the nucleic acidmolecules using a heater in the cartridge drive.

The cylindrical insert 302 is rotated to align the plunger with a portthat is in fluid communication with column chamber 318. When the plungeris activated, the solution is injected into of column chamber 318. Thesolution passes through a gel within the column chamber 318 andaccumulates in overflow chamber 324 of the column chamber 318. The gelmay be any suitable porous material, such as silica, that is useful forcleaning the solution. For example, column chamber 318 may be used toremove the nanoparticles or desalt the solution. The gel within columnchamber 318 may initially be in a dehydrated state. Prior to theinjection of the nucleic acid solution, water, buffers, or othersolutions may be withdrawn from other chambers and injected into columnchamber 318 through port 318P to hydrate the gel. Residual material maybe washed out of the column chamber 318 and into overflow chamber 324 bywithdrawing a wash solution from another chamber and passing the washsolution through the gel.

After the nanoparticles have been removed, the fee nucleic acids may besubjected to PCR. In one embodiment, PCR reagents are stored in adehydrated state. Like the gel of column chamber 318, water, buffers, orother solutions may be withdrawn from other chambers and injected intothe chamber which holds the PCR reagents to hydrate the reagents. Forexample, dehydrated PCR reagents may be stored in chamber 512 and watermay be stored in chamber 514. By rotating the cylindrical insert 302 andoperating the plunger, water is withdrawn from chamber 514, injectedinto chamber 512. The hydrated PCR reagents are then combined with thenucleic acid solution by, for example, injecting the nucleic acidsolution into chamber 512. The combined solution is then injected intoelongated chamber 404 (see FIG. 28A) that traverses at least a portionof a bottom edge of the cylindrical insert 302. The elongated chamber404 is configured to run a PCR process to amplify the concentration ofnucleic acids. In another embodiment, half the combined solution isinjected into elongated chamber 406 a and the other half of the solutionis injected into elongated chamber 406 b.

Elongated chamber 404 is similar to elongated chamber 402 describedelsewhere in this specification. The elongated chamber includes twozones that are sufficiently distant from one another such that each zonecan be placed over two different heaters that are at two differenttemperatures. A high temperature heater may be held at an elevatedtemperature (e.g. 90-95° C.) to break the hydrogen bonds in the nucleicacid sample that is disposed proximate that zone. However, thesetemperatures are too high for the PRC reagents to function. The lowtemperature heater may be held at an elevated temperature (e.g. 50-55°C.) that is below the high temperature heater but is above roomtemperature. These temperatures are too low to break the hydrogen bondsin the nucleic acid sample. However, these temperature are sufficientfor the PRC reagents to function. By rotating the cylindrical insert302, the two ends of the elongated chamber 404 can be sequentially sentthrough multiple high/low temperature cycles. For example, this cyclemay be repeated about thirty times.

In some embodiments, the nucleic acids are removed from the disposablecartridge and provided to external equipment for subsequent processing.In certain of these embodiments, the nucleic acids are stored inarchiving chamber 516 until they are ready to be removed from thedisposable cartridge.

In other embodiments, the nucleic acids remain within the disposablecartridge and are subjected to subsequent detection techniques toidentify the presence of absence of a target analyte. In such anembodiment, the amplified solution is withdrawn from the elongatedchannel 402 and subsequently aligned with a port that is in fluidcommunication with a reaction chamber, such as reaction chamber 142. Theplunger is activated and the amplified solution is injected intoreaction chamber 142. The reaction chamber 142 comprises a chip fordetecting the presence of particular nucleic acid sequences. Exemplarychips are disclosed in U.S. Pat. No. 6,399,303. The catalyst solutions,washing solutions and developer solutions necessary to permit the chipto detect the particular nucleic acid sequence are stored in otherchambers. These chambers are accessed in the same rotary fashion as theother chambers.

System for Preparing Nucleic Acid Samples:

Referring to FIG. 31A a system for preparing a nucleic acid sample isshown. The system 600 comprises a detection device 602 and a disposablecartridge 604. The disposable cartridge 604 removably attaches to acartridge drive 606 which is configured to rotate a cylindrical insertthat is rotatably connected to the disposable cartridge 604. When thedisposable cartridge 604 is properly positioned within detection device602 a plunger, which is operated by a plunger driver, aligns withsyringe mold 608. Additionally, a chip on disposable cartridge 604electrically connects to a chip receptacle in the detection device 602.This chip receptacle places the chip in electrical communication with amicroprocessor in the detection device 602 such that electrical signalsfrom the chip can be processed to detect the presence of a targetanalyte. Data may be stored on data storage media in the detectiondevice 602. Examples of data storage media include hard drives, flashmemory drives, and the like.

In one embodiment the disposable cartridge 604 includes a barcode labelthat encodes the specific disposable cartridge with identifyinginformation. This information includes, for example, informationconcerning the identification of the chip on that particular disposablecartridge. Manufacturing information, such as manufacturing location,lot number, and the like may also be included. In one embodiment, aunique identifier is provided in the barcode that permits the disposablecartridge to be specifically correlated with a particular test (e.g.test for disease X, on date Y for patient Z). The barcode may be aone-dimensional or two-dimensional barcode.

The detection device 602 may comprise a barcode scanner positioned toread the barcode on the disposable cartridge 604. This information maybe used by the microprocessor. For example, the barcode scanner may reada barcode on a particular disposable cartridge and determine thisdisposable cartridge is for testing for condition X. The detectiondevice 602 may display on screen 610 a message asking the user toconfirm condition X is the intended test. Additionally or alternatively,the detection device may detect that this particular disposablecartridge has already been used by querying a database for the uniqueidentifier associated with that disposable cartridge. In someembodiments, the previous test results are then loaded.

In the embodiments of FIG. 31B and FIG. 31C, two portable detectiondevices 616, 618 are shown. The portable detection devices are sized topermit an individual to transport the device into, for example, a fieldcondition. Such a portable detection devices are particularly useful inremote locations and find particular utility in military applications. Alid 612 opens to reveal a cartridge drive for receiving a disposablecartridge. A touch screen 614 provides a display and a user-interface.In other embodiments, a keyboard or button control is provided as auser-interface.

In the embodiment of FIG. 32 a bench-top detection device 700 is shown.The single detection device is configured to receive multiple disposablecartridges, each under a lid 612 a-f. A light 702 is provided for eachlid that indicates when a test is completed and the receptacle is readyfor use. For example, a red light may indicate the chamber is in usewhile a green light indicates the test is complete.

In one embodiment, the detection device, such as detection device 600,616, 618, or 700, can be connected to a computer network. In one suchembodiment, this connection is a wireless connection. The data obtainedmay be transmitted over the computer network to a server for subsequentprocessing. For example, the data obtained, including the positive ornegative detection of the analyte, the unique identifier of thedisposable cartridge, the date and time, as well as other pertinentinformation, may be sent to a server. In one embodiment, the detectiondevice is equipped with a global positioning system (GPS) and thegeographic location of the detection device is transmitted as well.Advantageously, this permits a server to compile data from one or moredetection devices and analyze the data as a function of both time andgeography. This feature is particularly useful when used in conjunctionwith field detection devices such as 616 and 618. Since this informationcan be transmitted with no user intervention, compliance with datatransmission protocols is increased. In certain embodiment, the data isstored in the data storage media until such time as the detection devicecan successful connect to the network. When a successful connection isestablished, the accumulated data is sent to the server.

Sample Types Processed

Numerous types of biological samples can be processed. The samplepreparation process is suitable for use on liquids, solids, soilsamples, animal tissue, insect carcasses, DNA, bacterial cells, sporesand viruses. Biological samples include all biological organisms whichcontain nucleic acids. Including but not limited to bacteria, spores,blood, tissues, fungi, plants and insects. As shown in FIG. 35, severaldisparate samples were processed using identical parameters. Samples ofpurified DNA, bacterial cells, spores, viruses and fruit flies were alltreated using the following technique: each sample was subjected tosonication treatment for two minutes in the presence of magneticnanoparticles and 100 micron glass beads. As shown in FIG. 35, allsample types provided a similar fragment distribution.

As a variety of types of biological samples can be used, a single systemcan be used with a wide variety of target organisms without the need tomodify the preparation process. Furthermore, even if a sample containstwo different targets, nucleic acid molecules can be purified from bothcomponents. For example, standard procedures may not work with a samplecontaining both a virus and a spore—either the parameters must be set toefficiently lyse the spores, in which case viral material is lost, orset to maximize the viral sample, in which case the spores are notlysed. Thus the benefits of the inclusion of a size stabilizer isevident.

By utilizing a single sample preparation technique the potential forfalse negatives is reduced. As the size stabilizer limits the range ofbase pair lengths for the nucleic acid molecules, the potential formaterial loss due to over-sonication is decreased. In one embodiment,the sample preparation system works with small quantities and produces anarrow distribution of nucleic acid molecule fragments. In oneembodiment, the preparation system passes sample through steps thatfilter the sample prior to applying a shear force.

Sample Disruption

In one embodiment the mechanical force used to release the nucleic acidmolecules is sonic vibration accomplished by contacting a container ofthe fragments suspended in protective buffer with source of sonicvibrations. Such a source may be a commercial ultrasonic transducer or apiezo electric crystal activated by an AC voltage. Such devices are wellknown to those skilled in the art. Shearing frequencies can be from10,000 Hz to 10 MHz. In one embodiment, the frequency is between 20 KHzand 4 MHz. In another embodiment, the frequency is between 20 KHz and 40KHz. To assist the shearing of protected nucleic acid molecules samplessuch as, for example, spores, small beads may be added to the sample.The sonic induced movement of the beads breaks the spore walls torelease the nucleic acid molecules contained within. The beads may rangein size from about 1 micron to about 1 mm. In one embodiment, the sizeis between about 10 microns to about 500 microns. In another embodiment,the size is between about 50 microns to about 200 microns. The beads maybe a metal such as stainless steel, glass or a dense metallic oxide suchas zirconium oxide. The time required for shearing the nucleic acidmolecules depends partly on the size of the sample and power transmittedfrom the transducer to the sample. However, when the sheared samplereaches a steady state, which depends on the composition of theprotective buffer, there is no further change in the nucleic acidmolecules size distribution with further sonication. In practice,sonication times of 15 seconds to 2 minutes at a power level of 1 to 2watts with a sample size of 100 μL of buffer containing 1 microgram ofnucleic acid molecules are sufficient to reach a steady state.

In one embodiment, disrupting beads such as glass beads of about 100microns in diameter are used to disrupt a sample and release nucleicacid molecules. The beads are vibrated using an ultrasonic source togenerate a shearing force on the sample. In one embodiment, for samplesuspensions from about 0.1 ml to 0.5 ml of water, containing from about0.1% to 1% nucleic acid, an ultrasonic power level of about 3 to 7 wattsis used for a period of from about 1 to 3 minutes. The volume of glassbeads used in the sample is, in one embodiment, between about 10% to 50%of the volume of the total suspension. The ultrasonic frequency used toagitate the glass beads is conventionally 20 KHz, from a commercialdevice such as the Branson Sonifier 150. It is understood thatfrequencies from about 10 KHz to 100 KHz could be suitable depending onthe sample parameters. In another embodiment, the shearing force isapplied by a nebulizer or a homogenizer.

FIG. 40 demonstrates the effective release of nucleic acid moleculesfrom spore samples. To determine efficiency of spore lysis, the maximumamount of nucleic acid output expected from the spores was estimated andcompared to the amount measured on the gel in FIG. 40. Utilizing thistechnique, the method provided an estimate of 85-90% efficiency.Alternatively, spore lysis efficiency can be measured by determiningspore survival after sonication. As shown in Table 1, based uponsurvival assays, the efficiency after two minutes of sonication duringexperiments was 86% of spores were opened.

Efficiency of Spore Lysis as Determined by Spore Survival (Spore Basis)

TABLE 1 Sonication time # spores survived % efficiency No sonication 23530 sec. 105 55%  1 min. 61 74%  2 min. 32 86%

For mechanical shearing such as bead disruption to be used as auniversal sample preparation approach, it is necessary to characterizeand optimize operating parameters with respect to different targetmaterial (DNA, RNA or protein) and their source (environmental, blood,or tissue). Although a single system is suitable for disruptiondifferent sample types, to optimize results parameters such as powerinput and the duration of applying sonic agitation may vary with respectto different cell types. Furthermore, it is understood that theconcentration of the size stabilizer, the size of the glass beads andthe inclusion of enzymes such as collagenase and hyaluronase are allfurther embodiments of the invention and are no way limiting.

It is understood that magnetic nanoparticles, glass beads or acombination of both can be used for disruption without departing fromthe invention. In one embodiment the magnetic nanoparticles are formedof iron oxides. In one embodiment the magnetic nanoparticles are in the40-200 nm size range. The magnetic nanoparticles can be acceleratedusing an ultrasonic force and can shred the sample. In one embodiment,glass beads are used in the extraction mixture for efficient lysis ofspores.

In another embodiment, the sample preparation process further includesthe addition of RNase inhibitors to prevent sample degradation. In oneembodiment, the sample preparation process includes diethylpyrocarbonate(DEPC), ethylene diamine tetraacetic acid (EDTA), proteinase K, or acombination thereof.

Size Stabilizers

In one embodiment, a buffer is mixed with the biological sample duringthe disruption step. To retain the desired sample size the buffer servesas a size stabilizer. The size stabilizer is a water solution which maycontain salts, detergents, co-solvents or polymers. The size stabilizerprevents the subsequent shearing step from producing fragments ofnucleic acid molecules that are too small to be useful in operationssuch as hybridization, sequencing and polymerase chain reaction (PCR)amplification. For hybridization, fragments of nucleic acid moleculesthat are smaller than about 18 base pairs lose specificity and areunstable at ambient temperatures. For genetic sequencing and PCRapplications, nucleic acid molecule fragments from about 200 to about500 base pairs are desirable. Use of a pure water buffer gives nucleicacid molecule fragments less than about 100 base pairs, which are toosmall for many applications.

The addition of size stabilizers in the sample preparation of thisinvention results in a high yield of nucleic acids of limited sizerange. The size stabilizers of this invention include detergents,surfactants and soaps. Examples of suitable stabilizers include anionicsurfactants, sodium dodecylsulfate, and sodium dodecylbenzenesulfonate.The size stabilizer is present in the sonicated suspension in an amountbetween about 0.1% and 10%. In another embodiment, the size stabilizersis present in an amount between about 0.2% and 2%. In yet anotherembodiment, the size stabilizers is present an amount between about 0.5and 1.5%.

Use of the size stabilizer allows the gathering of nucleic acid moleculefragments in a desired base pair range. In traditional bead beatingprocesses the mechanical shearing force is turned off after a particulartime to maximize the amount of nucleic acid molecule fragments in thedesired base pair range. However, because the process is time sensitivea large range of base pair lengths remain present in the sample. Byutilizing a size stabilizer the base pair length of most of the samplecan be fragmented to the desired base pair range. In one embodiment, atleast 60% of the nucleic acid molecule fragments are within 50% of thelength of the median nucleic acid molecule fragment base pair length inthe sample. Said another way, if the median nucleic acid moleculefragment has 400 base pairs, 60% of the sample would have between 200and 600 base pairs. In another embodiment, at least 75% of the nucleicacid molecule fragments are within 50% of the length of the mediannucleic acid molecule fragment base pair length in the sample. In yetanother embodiment, at least 75% of the nucleic acid molecule fragmentsare within 30% of the length of the median nucleic acid moleculefragment base pair length in the sample.

Without a size stabilizer present, the nucleic acid molecules tend todegrade when applying a mechanical force such as sonication. Theultrasonic bead beating with a size stabilizer present shears thenucleic acid molecules into short fragments that are less than 100 baseslong (See FIG. 34, lanes 5 and 6). For most applications, fragments needto be larger than 100 bases. As shown in FIG. 42, a series of tests wereperformed to sonicate purified DNA and RNA sheared polymers to nosmaller than 400 bases, even under lengthy sonication times. In complexsamples, nucleic acid molecules stick to membranes and proteins whilecontinuing to break down to smaller fragments. To overcome this problem,the lysis buffer is modified to contain a size stabilizer such as adetergent like sodium dodecyl sulfate (SDS). As shown in FIG. 34, theaddition of the size stabilizer shown in lanes 3 and 4 protects thenucleic acid molecules from over shearing. The samples without the sizestabilizer were sheared to well below 100 bases, as shown in lanes 5 and6.

The size stabilizer is contained in a protective buffer solution. It isunderstood that the protective buffer may contain numerous sizestabilizers to achieve the desired base pair range. Salts which may beused in the protective buffer include, sodium phosphate, guanidiniumhydrochloride and dextran sulfate. The protective buffer may furthercontain detergents such as sodium dodecyl sulfate, sodium dodceylbenzene sulfate, and polyethyleneglycol. Many commercial anionicsurfactants such as ALKANOL® XC may also be used. In another embodimentthe protective buffer includes co-solvents. Co-solvents include dipoleaprotic solvents such as dimethylsulfoxide, dimethyl formamide, dimethylacetamide, hexamethyl phosphoramide and tetramethylurea. In anotherembodiment the protective solution contains polymers such as poly vinylalcohol, polyethylenimine, poly acrylic acid and other polymeric acids.The concentration of the salts, detergents, co-solvents and polymers mayrange from 10 mM to 5M. In one embodiment, the concentration is betweenabout 100 mM to about 1M. Other size stabilizers of this inventioninclude chaotropic salts such as guanadium thiocyanate. Such salts areknown to disrupt the normal folding of proteins associated with nucleicacids, thereby releasing the nucleic acids in free form.

In another embodiment, the presence of a size stabilizer also stabilizesRNA. The SDS and guandinium thiocyanate disrupt the RNAses in the samplethus preserving the RNA.

Cleaning of Fragmented Nucleic Acids

In one embodiment, the process further comprises the steps necessary toclean the nucleic acid molecules. After release of the nucleic acidmolecules and shearing to a useful size range, it is advantageous toclean the nucleic acid molecules from cell debris, proteins, sonicationbeads and the protection buffer to provide a purified nucleic acidmolecule solution in a buffer compatible with subsequent nucleic acidmolecule operations and procedures.

In one embodiment, additional rinse steps are used to purify the sample.The rinsing removes compounds which could inhibit binding of nucleicacid molecules. Suitable rinse solutions include, but are not limited toalcohol solutions such as ethanol. The sample can be washed withadditional precipitation buffer, or a washing buffer that does notdisturb the complex. After washing, the buffer is drained from thesample resulting in a purified, concentrated sample.

In one embodiment, the nucleic acid molecules are cleaned bymagnetically separating them from the reminder of the sample. Thenucleic acid molecules bind to magnetic nanoparticles. In oneembodiment, the binding occurs in a high salt/alcohol condition and thenucleic acid molecules are eluted using a low salt chelating buffer suchas sodium citrate at increased temperature. In one embodiment the sampleis heated to at least 60° C. to increase the yield from elution.

Once the magnetic nanoparticles are attached to the nucleic acids amagnetic field is applied to the reaction chamber. The application ofthe magnetic field causes the magnetic nanoparticles and any attachedtarget analytes to concentrate in one portion of the reaction chamber.The sample is pulled from the concentrated region of the sample chamberproviding a large amount of target analytes compared to the amount ofvolume extracted. By concentrating the sample more sensitive tests canbe preformed.

In another embodiment, the magnetic field holds the magneticnanoparticle steady as the remaining sample is removed from the chamber.The binding force between the magnetic nanoparticle and the targetanalyte is sufficient to prevent the target analyte from being removed.A magnet is utilized to generate an magnetic field. The magnet can pullor push magnetic nanoparticles. The magnet can concentrate a sample ofmagnetic nanoparticles or speed up the diffusion process by guiding anymagnetic nanoparticles.

In one embodiment, magnetic nanoparticles are located in a samplechamber along with a target analyte. The magnetic nanoparticles have anaffinity for the target analyte. By attaching the magnetic nanoparticlesto the target analyte and applying a magnetic field the target analyteis manipulated to desired locations within the sample chamber.

In one embodiment, a precipitation buffer is in solution with the targetanalyte fragments and the magnetic nanoparticle. The precipitationbuffer precipitates the target analyte out of solution and the targetanalyte is drawn to the magnetic nanoparticles. The precipitation buffercan be any buffer that precipitates the target analyte from thesolution. For proteins, examples of suitable precipitation buffersinclude, but are not limited to organic precipitants such as, ammoniumsulfate, trichloroacetic acid, acetone, or a mixture of chloroform andmethanol. For nucleic acid molecules suitable precipitation buffersinclude, but are not limited to, water miscible organic solvents,acetone, dioxane and tetrahydrofuran. While examples of precipitationbuffers are provided, it is understood that any suitable precipitationbuffer can be utilized without deflecting from this claimed invention.

In one embodiment a dispersion of magnetic nanoparticles is added to thesample. The mixture is then incubated at about 60° C. to facilitate thebinding. A precipitation buffer is then added to the mixture. The boundcomplex of nucleic acid molecules and magnetite is then collected in amagnetic field. In one embodiment, the complex is collected on a sidewall of the container so any unbound solids can fall to the bottom ofthe container for easy removal. The buffer and any unbound solids arethen removed from the sample.

For further processing of the nucleic acid molecules, for someprocesses, it is necessary to remove the magnetite nanoparticles. In oneembodiment the nucleic acid molecule is eluted from the complex ofnucleic acid molecules and magnetite by heating a mixture of an elutionbuffer and the complex to 95° C. The magnetite can be collected by amagnetic field, or by centrifugation, providing purified nucleic acidmolecules in elution buffer. In one embodiment the elution bufferscontain a salt which interacts strongly with iron oxide surfaces. In oneembodiment, the buffers are selected from phosphate and citrate saltsolutions.

In another embodiment, the magnetic nanoparticles containsuperparamagnetic nanoparticles. The superparamagnetic nanoparticlesinclude metal oxides, such as iron oxides. In one embodiment themagnetic nanoparticle is a magnetite nanoparticle (Fe₃O₄). Magnetiteparticles are common in nature, and can be collected from beach sands atthe edge of the ocean by screening with a magnet. Grinding theseparticles will produce a relatively coarse magnetic powder. Smallersized particles can be produced by adding a solution of mixed ferric andferrous chloride to a stirred aqueous alkaline solution of sodium orammonium hydroxide. Even smaller sized particles are produced by thermaldecomposition of iron acetonylacetonate in dibenzyl ether in thepresence of hexadecanediol, oleyl amine and oleic acid. Numerous methodsfor making magnetite are known. For example, Sun et al. discloses slowlyadding a mixture of ferric and ferrous chloride into stirred ammonia.Langmuir, 2009, 25 (10), pp 5969-5973. U.S. Pat. No. 4,698,302 teachesmixing ferrous and ferric chloride with sodium hydroxide. Samanta et al,discloses adding ammonia to a stirred mixture of ferric and ferrouschloride in an inert atmosphere. Journal of Materials Chemistry, 2008,18, 1204-1208. Duan et al. teaches dissolving iron oxide in oleic acidto form a complex that forms magnetite nanoparticles when heated to 300degrees C. J. Phys. nucleic acid molecule Chem. C, 2008, 112 (22), pp8127-8131. Additionally, Yin et al. discloses thermally decomposing ironpentacarbonyl in the presence of oleic acid, Journal of MaterialsResearch, 2004, 19, 1208-1215.

Suitable binding buffers may be added to the solution. Binding buffersfor the nucleic acid molecule/magnetite complex are, for the most part,buffers in which nucleic acid molecules are insoluble. Precipitation ofthe nucleic acid molecules promotes binding of the nucleic acidmolecules to the magnetite nanoparticles. The binding buffer for nucleicacid molecules and magnetite nanoparticles may contain water, sodiumacetate, sodium chloride, lithium chloride, ammonium acetate, magnesiumchloride, ethanol, propanol, butanol, glycogen or other sugars,polyacrylamide or mixtures thereof. In one embodiment the binding bufferis isopropanol.

Binding of the nucleic acid molecules to the magnetite nanoparticles isnot instantaneous. In one embodiment the mixture is incubated above roomtemperature to speed the binding process.

Magnetic Manipulation:

In one embodiment, a magnet 114 is utilized to generate an electricfield. The magnet can pull or push magnetic nanoparticles in thecylindrical insert. The magnet 114 can concentrate a sample of magneticnanoparticles or speed up the diffusion process by guiding any magneticnanoparticles.

Magnetic nanoparticles are located in a sample chamber along with atarget analyte (e.g. a target nucleic acid). The magnetic nanoparticleshave an affinity for the target analyte. By attaching the magneticnanoparticles to the target analyte and applying a magnetic field thetarget analyte is manipulated to desired locations within the samplechamber.

In one embodiment, the target analyte binding element is attached to themagnetic nanoparticle via at least one intermediate connecting groupsuch as, but not limited to linkers, scaffolds, stabilizers or stericstabilizers.

The magnetic nanoparticles exhibit magnetic properties. In oneembodiment cobalt, nickel, iron or a combination thereof is used tocreate a magnetic nanoparticle. In one embodiment, the magneticnanoparticle further contains a catalytic particle. In one embodimentthe catalytic particle is palladium, platinum, silver or gold.

In one form, a nickel-palladium nanoparticle, stabilized by a surfacelayer of 4-dimethylaminopyridine as described in Flanagan et al,Langmuir, 2007, 23, 12508-12520, is treated by adsorption with aplurality of ethidium bromide intercalator molecules to create nucleicacid binding sites. The ethidium moiety bonds to the nucleic acidpolymer thereby attaching the nickel-palladium nanoparticle to thenucleic acid polymer.

In another form, a simple straight-chain scaffold molecule, such asoligoethylene glycol (PEG), is affixed with a nucleic acid bindingelement at one end and a linker at the other end. The nucleic acidbinding element binds to the nucleic acid polymer and the linker bindsto the magnetic nanoparticle. The nucleic acid binding element is anintercalator, such as ethidium bromide, or a minor groove binder such asdistamycin. The linker is a phenanthroline derivative. Hainfeld, J.Structural Biology, 127, 177-184 (1999) reports the advantage ofphenanthroline derivatives in creating palladium particles. The scaffoldmay be a simple difunctional straight chain as shown, or may be amultifunctional branched scaffold connecting multiple magneticnanoparticles or nucleic acid binding elements. The nucleic acid bindingelement bonds to the nucleic acid polymer, thereby attaching thenanoparticle to the nucleic acid polymer. It is understood thatadditional nucleic acid binding elements and intermediate connectinggroups are within the scope and may be used.

Concentration of Target Analyte:

The sample containing the target analyte is located in a reactionchamber. The reaction chamber contains both the sample and magneticnanoparticles. The magnetic nanoparticles bind to the target analyte. Inone embodiment the reaction chamber further contains disrupting beads toassist in breaking apart samples to provide access to the targetanalyte.

Once the nucleic acid molecules have been released, the nucleic acidmolecules can be magnetically separated from the reminder of the sample.The nucleic acid molecules bind to magnetic nanoparticles. In oneembodiment, the binding occurs in a high salt/alcohol condition to forma complex. The complex is eluted using a low salt chelating buffer suchas sodium citrate with increased temperature. In one embodiment thecomplex is heated to at least 95° C. to increase the yield from elution.

Once the magnetic nanoparticles are attached to the target analyte amagnetic field is applied to the reaction chamber. The application ofthe magnetic field causes the magnetic nanoparticles and any attachedtarget analytes to concentrate in one portion of the reaction chamber.The sample is pulled from the concentrated region of the sample chamberproviding a large amount of target analytes comparative the amount ofvolume extracted. By concentrating the sample more sensitive tests canbe preformed.

In another embodiment, the magnetic field holds the magneticnanoparticle steady as the remaining sample is removed from the chamber.The binding force between the magnetic nanoparticle and the targetanalyte is sufficient to prevent the target analyte from being removed.In some embodiments, additional rinse steps are used to purify thesample.

Rapid Movement and Increased Sensitivity:

Typically in solution a target analyte is limited in movement by fluidflow and diffusion rates. To speed the movement of a target analytethrough the system a magnetic field is applied to progress the magneticnanoparticle to the desired location. The application of the magneticfield allows for rapid transport of the target analyte from one chamberto another.

An array of sensors are used to rapidly detect the target analyte. Amagnetic field is applied to guide the magnetic nanoparticles andattached analytes to the vicinity of a first sensor. A distinct magneticfield then guides the magnetic nanoparticles and any attached targetanalytes to a second sensor. The magnetic field is manipulated to movethe target analytes to each sensor in the array. In one embodiment, thesensor binds a particular target analyte with enough force to preventthe magnetic field from breaking the bond. By systematically applyingmagnetic fields the analysis time is greatly reduced compared to normaldiffusion analysis.

Magnetic Nanoparticles:

Use of sols or clusters in the form of magnetic nanoparticles allows forthe attachment of magnetic material to a target nucleic acid polymer orother target analyte. By applying a magnetic field to the sample thenucleic acid polymer can be manipulated via the attached paramagnetmaterial.

The paramagnet nanoparticles are formed in solution with a stabilizer.In one embodiment a metal salt is used. A reducing agent, such asdimethylamineborane or sodium borohydride, is added to the solution. Ifneeded, solvents and excess salts can be removed by centrifugation,decantation, washing, and resuspension of the metal clusters.Alternatively, a magnetic field can be applied to the solution holdingthe magnetic nanoparticles in place as a drain and rinse is applied.

Target Analyte Binding Element:

The target analyte binding element attaches to the magneticnanoparticle, either directly or by way of an intermediate connectinggroup. The target analyte binding element further binds to the nucleicacid polymer. In one embodiment the target analyte binding element is anucleic acid binding element such as a molecule, fragment or functionalgroup that binds to nucleic acid polymers. Potential nucleic acidbinding elements comprise intercalators, minor groove binders, cations,amine reactive groups such as aldehydes and alkylating agents, proteins,and association with hydrophobic groups of surfactants. In addition,functional groups such as aldehydes are used to create a connection byreaction with free amines in the nucleic acid. Other amine reactivegroups such as electrophiles for use in Michael addition reactions aresuitable.

Examples of structures that form the basis for intercalating and minorgroove binder structures are:

The range of specific intercalator and minor groove binder structures isenormous as the field has been the subject of intense study for over 50years. See R. Martinez and L Chacon-Garcia, Current Medicinal Chemistry,2005, 12, 127-151. Therefore, the R groups include a broad range oforganic functional groups. In many cases, interaction can be enhanced ifR contains hydrogen bonding, cationic or hydrophilic character.

In addition, compounds such as cationic polymers, such aspolyethyleneimine, interact with nucleic acid and have been proposed asgene carriers as evidenced by Xu et al, International Journal ofNanoscience, 2006, 5, 753-756 and Petersen et al, BioconjugateChemistry, 2002, 13, 845-854. Proteins are another well known class ofmaterials that offer useful nucleic acid interaction and can be thebasis for attaching nanoparticles to nucleic acids. Direct reaction withfunctional groups on the nucleic acid is also within the scope of thisinvention. For example, amine groups can be reacted with aldehydes tocreate a bond (Braun et al, Nano Letters, 2004, 4, 323-326)

In one embodiment the nucleic acid binding elements are specific bindingagents that specifically target double-stranded nucleic acid moleculeswhile not binding with single-stranded nucleic acid molecules. Forexample, minor-groove binding compounds specifically bind hybridizeddouble-stranded DNA molecules, but do not bind to single-strandedoligonucleotide capture probes. In contrast, palladium chloride reagentindiscriminately binds to both the target molecules and capture probes.The binding element binds specifically to the target nucleic acidmolecule while having little or no affinity towards non-targetmolecules. It is understood that the specific binding elements caninclude but are not limited to intercalators, minor-groove bindingcompounds, major-groove binding compounds, antibodies, and DNA bindingproteins. The specific binding element binds to a specific site on atarget nucleic acid without binding to non-desired areas. In oneembodiment, the specific binding element is ethidium bromide. Inalternative embodiments, the specific binding element is distamycin,idarubicin, or Hoescht dye.

In one embodiment the nucleic acid binding element also serves as astabilizer as described elsewhere in this specification.

Stabilizers:

In one embodiment, the magnetic nanoparticles are surface functionalizedwith stabilizers to impart desirable properties. These stabilizedmagnetic nanoparticles demonstrate colloid stability and minimalnon-specific binding. Furthermore, the presence of the stabilizer insolution while forming the magnetic nanoparticle controls thenanoparticle size.

The stabilizer provides colloid stability and prevents coagulation andsettling of the magnetic nanoparticle. The stabilizer further serves tolimit the size of the magnetic nanoparticle during the formationprocess. In one embodiment, metal magnetic nanoparticle are formed in asolution containing stabilizer and metal ions. In one embodiment thestabilizers are chelating compounds. Large magnetic nanoparticles areundesirable as they are more likely to precipitate out of solution.Therefore, the magnetic nanoparticle shall be small enough to remain insolution. In one embodiment, the magnetic nanoparticle is generallyspherical in shape with a diameter from about 0.5-1000 nm. In oneembodiment, the magnetic nanoparticle is generally spherical in shapeand has a diameter from about 1-100 nm.

Suitable stabilizers include, but are not limited to,polyethyloxazoline, polyvinylpyrollidinone, polyethyleneimine,polyvinylalcohol, polyethyleneglycol, polyester ionomers, silicone ionicpolymers, ionic polymers, copolymers, starches, gum Arabic, surfactants,nonionic surfactants, ionic surfactants, fluorocarbon containingsurfactants and sugars. In one embodiment the stabilizer is aphenanthroline, bipyridine and oligovinylpyridine of the followingformulas:

where R₁ is COOH, CH₂OH, CH₂NH₂, or CH₂NHCH₃; and

-   -   R₂ is H, COOH, CH₂OH, CH₂NH₂, NH or CH₂NHCH₃.

In one embodiment where the magnetic nanoparticle contains palladium,these stabilizers link by acting as ligands for palladium ions and aretherefore closely associated with the particle formation. In addition tolinking, the stabilizers have hydrophilic groups that interact with thewater phase. The linking and stabilization function of molecules such asphenathrolines in palladium particle formation is further described inHainfeld, J. Structural Biology, 127, 177-184 (1999).

It is understood that particles derived from a broad class of materials(plastics, pigments, oils, etc) in water can be stabilized by a widearray of surfactants and dispersants that don't rely on specificcoordination. These classes of stabilizers are also within the scope ofthis invention.

In one embodiment the stabilizer stabilizes the magnetic nanoparticlefrom precipitation, coagulation and minimizes the non-specific bindingto random surfaces. In another embodiment, the stabilizer furtherfunctions as a nucleic acid binding element as described below.

Linker:

The linker is bound directly to the magnetic nanoparticle to allow theattachment of other intermediate connecting groups or target analytebinding elements. It is understood that the linker can also serve as astabilizer or scaffold.

The linker can be bound through various binding energies. The totalbinding energy consists of the sum of all the covalent, ionic, entropic,Van der Walls and any other forces binding the linker to the magneticnanoparticle. In one embodiment, the total binding energy between thelinker and the magnetic nanoparticle is greater than about 10 kJ/mole.In another embodiment the total binding energy between the linker andthe magnetic nanoparticle is greater than about 40 kJ/mole. Suitablelinkers include, but are not limited to ligands, phenanthrolines,bidentates, tridentates, bipyridines, pyridines, tripyridines,polyvinylpyridines, porphyrins, disulfides, amine acetoacetates, amines,thiols, acids, alcohols and hydrophobic groups.

Scaffold Compositions:

The magnetic acid binding element may be connected directly to themagnetic nanoparticle or a linker. Alternatively, the nucleic acidbinding element is attached to a scaffold, either individually or as amultiplicity. In either case, the final conjugate is endowed with thetwo essential properties—nucleic acid specific recognition-binding andan attached magnetic nanoparticle. Attaching the nucleic acid bindingelement to the scaffold may be by way of any of the common organicbonding groups such as esters, amides and the like.

Attachment to a common scaffold creates an enormous range of possiblesizes, shapes, architectures and additional functions. In one embodimentthe scaffold composition is a linear chain with the two functionalgroups at the ends. The chain itself can be of any composition, lengthand ionic character. In an alternative embodiment, often used inbiological applications, polyethylene glycol with a reactive amine, acidor alcohol end groups is utilized as included in the following example.

Linear short spacers with cationic character can be desirable as theycan enhance intercalation performance.

A polymeric or oligomeric scaffold allows for multiple groups to bejoined in the same structure where the number of groups is limited onlyby the size of the chain.

In addition to short and long chain structures scaffolds can be builtwith branched or very highly branched architectures. Furthermore,scaffolds can be a microgel particle with nanoparticles bound to aswollen polyvinylpyridine interior and peripheral nucleic acid bindingelements are illustrated. In another embodiment the scaffold is acore-shell latex particle with magnetic nanoparticles centers andperipheral nucleic acid recognition groups populating the surface. It isunderstood that any scaffold compositions can be incorporated to connectintermediate connecting groups, magnetic nanoparticles or nucleic acidbinding elements.

Steric Stabilizers:

In one embodiment a steric stabilizer is used to attach the targetanalyte binding element to the magnetic nanoparticle. The stericstabilizer is capable of functioning as a stabilizer, linker andscaffold as described above. In one embodiment the steric stabilizer ispolyethylenimine, polyethyloxazoline or polyvinylpyrrolidone. The stericstabilizer binds to the magnetic nanoparticle with a total bindingenergy of at least 10 kJ/mole. In another embodiment the stericstabilizer binds to the magnetic nanoparticle with a total bindingenergy of at least 40 kJ/mole. The use of steric stabilizers eliminateany need for distinct stabilizers, linkers, or scaffolds. One ormultiple nucleic acid binding elements can be attached to the stericstabilizer. Furthermore, one or multiple magnetic nanoparticles can bebound to the steric stabilizer.

Target Analyte Binding Substance:

In one embodiment for forming the target analyte binding substance on amagnetic nanoparticle, the magnetic nanoparticles are formed in solutionwith a stabilizer such as dimethyaminopyridine (DMAP). The stabilizedmagnetic nanoparticles are purified to retain clusters of the desiredsize. The nanoparticles are then treated directly with a nucleic acidbinding element such as ethidium bromide or with a nucleic acid bindingelement connected to a linker or with a scaffold composition containingthe nucleic acid binding element. The scaffold composition can be apolymer containing nucleic acid binding elements such as naphthalimideor acridine. The polymer displaces some of the DMAP and attaches to theparticle. It is understood that the nucleic acid binding element can bechemically attached to the scaffold composition prior to the attachmentof the scaffold composition to the particle.

In another embodiment for forming the target analyte binding substanceon a magnetic nanoparticle, the magnetic nanoparticles are formed insolution in the presence of a nucleic acid binding element such asethidium bromide or in the presence of a nucleic acid binding elementconnected to a linker or in the presence of a scaffold compositioncontaining the nucleic acid binding element. The scaffold compositioncan be a polymer containing nucleic acid binding elements such asnaphthalimide or acridine. It is understood that the nucleic acidbinding substance connects to the particle during the particle formationprocess and may offer some colloidal stability to the dispersion. Inaddition, stabilizers in the form of ionic surfactants, non ionicsurfactants, water soluble oligomers and polymers may also be added toenhance colloid stability and control particle size.

In one embodiment, the nucleic acid molecules are used for PCRapplication after preparation. It is known that PCR applications do notwork successfully in the presence of detergents and alcohol. Therefore,for PCR application and additional filtering or cleaning step isutilized to prepare the sample prior to testing.

EXAMPLES Sonication Bead Disruption

Spores were prepared and isolated from Bacillus subtilis fromsporulation media+. To a 100 ul aliquot of the spores taken from theculture, an equal volume of 0.1 mm glass beads were added in a microfugetube. The tip of the microfuge tube was placed in the socket of aBranson Ultrasonic sonicator. Using a power setting of 2, the beadswithin the tube were agitated for two minutes. Afterwards, gram stainingshowed that greater than 90% of the spores were disrupted by thisprocess. This was confirmed with plating assays by counting coloniesformed from spores surviving the process. Estimation of the amount ofDNA released was accomplished by spotting an aliquot of the lysate ontothe surface of a 1% agarose gel containing 1 mg/ml ethidium bromide. ABio-Rad Fluor-S imager compared the intensity of the sample fluorescenceagainst known standard amounts of DNA also spotted onto the gel surface.Using this technique, approximately 10 ng of DNA can be isolated from2.5×10⁵ spores.

Magnetic Examples

Metal salts (nickel, cobalt, iron) with a small amount of palladium saltare dissolved in a solvent (water and/or polar organic solvent) alongwith a stabilizer (phenanthroline, bipyridine, polyvinylpyrrolidinone).A reducing agent is added (dimethylamineborane, sodium borohydride) andthe mixture is held until the metal clusters are formed. If needed,solvents and excess salts can be removed by centrifugation, decantation,washing, and resuspension of the metal clusters.

Solution A—24 g of nickel chloride hexahydrate and 44 g of sodiumcitrate were dissolved in 500 ml of water.

Solution B—24 g of ethanolamine were dissolved in 500 ml of water.

Solution C—5 g of cobalt chloride hexahydrate were dissolved in 100 mlwater.

Solution D—2 g of potassium tetrachloropallidate and 6 g of potassiumchloride were dissolved in 100 ml of water.

Solution E—1 g of bathophenanthroline-disulfonic acid, disodium salthydrate was dissolved in 100 ml water.

Solution F—3 g of dimethylamine borane were dissolved in 100 ml water.

Magnetic Example 1

In a 20 ml glass vial, 1 ml solution A and 1 ml of solution B weremixed. 0.1 ml of solution D was added, followed immediately by 0.2 ml ofsolution E. Then 0.5 ml of solution F was added and the mixture was heldat 60 degrees C. for 30 minutes. After cooling to room temperature, themixture was placed in a strong magnetic field for 10 seconds (themagnetic field was from the permanent magnetic removed from a discardedcomputer hard drive) and it was observed that most of the metal clustersmoved to the wall of the vial nearest the magnet.

Magnetic Example 2

In a 20 ml glass vial, 0.2 ml solution A, 0.8 ml solution C and 1 ml ofsolution B were mixed. 0.1 ml of solution D was added, followedimmediately by 0.2 ml of solution E. Then 0.5 ml of solution F was addedand the mixture was held at 60 degrees C. for 30 minutes. After coolingto room temperature, the mixture was placed in a strong magnetic fieldfor 10 seconds (the magnetic field was from the permanent magneticremoved from a discarded computer hard drive) and it was observed thatmost of the metal clusters moved to the wall of the vial nearest themagnet.

Preparation of Magnetite Clusters Example

A first solution of ferric chloride (0.8M), ferrous chloride (0.4M) andhydrochloric acid (0.4M) was mixed and 0.2 micron filtered. A secondsolution was prepared with 72 ml of ammonium hydroxide (30%) with waterto make 1 liter.

1 ml of the ferric/ferrous chloride solution was added with stirring to20 ml of the ammonium hydroxide solution. Stirring was continued for 15seconds. The solution (in a 20 ml vial) was placed on a strong magnetand allowed to stand for 1 minute, after which all the product waspulled to the bottom of the vial. The clear supernatant liquid wasdecanted, replaced with water, mixed, and placed near the magnet. Againthe product was pulled to the bottom of the vial. This process wasrepeated three times to wash the product free from any residual ammoniumand iron salts. The vial was then filled with 20 ml of water andultra-sonicated for 5 minutes at 4 watts power. The suspension was thenfiltered through a 1 micron glass filter to give a stable suspension ofmagnetite nanoparticles that remain in suspension until pulled down bymagnetic forces or centrifugation.

Attachment of Magnetic Nanoparticles Example

Nucleic acid molecules were purified from fruit flies, then lysed withferrite nanoparticles followed by magnetic separation and elution. Themagnetic beads captured more than 90% of available nucleic acidmolecules.

Hybridizing to Capture Probes Example

Once the nucleic acid molecules are prepared, they are hybridized tocapture probes on sensor electrodes. Samples of nucleic acid moleculesfrom Bacillus cells were prepared through ultrasonic lysis and magneticconcentration. The eluted DNA was bound to probes on the sensor chip todemonstrate that there are no inhibitors of hybridization.

Sample Cleaning:

In one embodiment, the sample is cleaned to remove compounds which couldpotentially inhibit the binding of nucleic acid molecules to sensors. Byattaching magnetic nanoparticles to the sample and manipulating thesample with a magnetic field the sample is both concentrated and cleanedfrom impurities.

Cleaning Example

Bacterial and spore samples mixed with soil were processed to evaluatecomplex samples. Soil is a complex medium which is known to inhibitPCR-based systems. Soil was added to samples containing six whole fruitflies. The flies are intended to represent insects that might beevaluated for carrying a disease like malaria. Up to 320 micrograms ofthe soil were added per milliliter of sample. The fruit flies were lysedand the DNA and RNA were captured using ferrite nanoparticles with theaddition of ethanol. The magnetic nanoparticles were collectedmagnetically, washed with buffer and ethanol to remove contaminants thenconcentrated with magnetics. The nucleic acid molecules were then elutedin hybridization buffer at 90° C. to denature the DNA component. Theferric nanoparticles worked well in the presence of soil. Minimal losswas seen until the level of soil in the sample reached 32 milligrams per100 micro liters where the solution becomes viscous and particlemovement is difficult.

DNA from Complex Samples Example

Bacillus cells were mixed with cattle ear tissue or whole fruit fliesand the mixtures were taken through the sample preparation process. Theresulting nucleic acids were hybridized to probes on sensor chips. Thechips were then treated with YOYO-1 dye to detect hybridized DNA. Thetarget DNA sequences in the cells hybridized to the sensor chips atlevels comparable to Bacillus cells processed separately. Negativecontrols without Bacillus showed no hybridized DNA. The experiment wasrepeated with dirt added to the samples as described above.Hybridization efficiency remained at least 60% of the hybridization seenin the sample without eukaryotic cells and dirt.

Washing Magnetic Nanoparticles with a Flow Example

Magnetic nanoparticles were bound to DNA and then the solutionintroduced into a clear plastic tube with a 2 mm diameter. A magnet wasplaced under the center of the tube. A wash buffer was pushed throughthe tube using a syringe pump. The magnetic nanoparticles visuallyremained in place through the washing. After washing the magnet wasremoved and the magnetic nanoparticles were rinsed out of the tube. DNAwas eluted at high temperature and run on a gel. No apparent loss of DNAwas observed.

Efficiency of Binding and Release of Magnetic Nanoparticles Example

Radiolabled DNA was used to determine the efficiency of binding toferrite and the release of the nucleic acid molecules. Radiolabeled DNAwith the magnetite suspension and three volumes of ethanol were mixed.The magnetite was pulled to the bottom of the tube using a magnet. Thesupernatant fluid was removed from the pellet and both fractions werecounted in a scintillation counter. Binding was measured as a functionof the fraction of ethanol in the mix. The results are shown in FIG. 36.

To determine the release efficiency, the bound DNA pellet is suspendedin 100 μL of buffer as indicated in the table below, incubated for 10minutes at 95° C., then collected on the magnet. The supernatant wasseparated from the pellet and both were counted.

Buffer Supernatant cpm Pellet cpm % Free 500 mM Phosphate 43,450 192596%  50 mM Phosphate 18,409 684 96%  60 mM Citrate 33,276 2164 94% 100mM Tris 911 35,878 3% 0.2% SDS 1.5% Dextran sulfate

The Tris buffer with SDS can be used for hybridization with magnetitebound DNA in order to allow for magnetic concentration of DNA or RNAnear the sensor.

Rapid Movement of Magnetic Nanoparticles Example

Microchips were fabricated with metal coils having line widths of onemicron. A current was run through the coils to produce a magnetic field.A solution containing magnetic nanoparticles was then spotted over thecoils. The chip was placed under a microscope and current turned onthrough the coil. Within 10 seconds, clusters were congregating at thecorners within the coil. Once the current was turned off the magneticnanoparticles demagnetize and begin to diffuse back into solution.

Tissue Samples

As shown in FIG. 37, for diagnostic samples, an approach using tissuefrom the ear of a cow was evaluated. Ear tissue is often taken fromcattle for evaluation and has skin, hair, large amounts of cartilage andis rich in blood. Ear plugs of about 3 mm in diameter were tested. Arobust sample of about 1 microgram of nucleic acid molecules wasisolated from an earplug using ultrasonication and 40 nm ferritenanoparticles. The nucleic acid molecules were in the expected sizerange. Glass beads were not required for extraction from the tissue andsubsequent treatment of an ear plug with bead beating did not result inadditional nucleic acid molecule extraction. Sonication power and timesettings were identical to those used in the previous examples.

Samples Contaminated with Soil

As shown in FIG. 38, to evaluate complex samples, bacterial and sporesamples mixed with soil were processed. Soil is a complex medium whichis known to inhibit PCR-based systems. Soil was added to samplescontaining six whole fruit flies. The flies are intended to representinsects that might be evaluated for carrying a disease like malaria. Upto 32 milligrams of the soil were added per milliliter of sample. Thefruit flies were disrupted using ultrasonication in the presence offerrite nanoparticles for two minutes. DNA and RNA were captured usingferrite nanoparticles with the addition of ethanol. The nanoparticleswere collected magnetically, washed with buffer and ethanol to removecontaminants then concentrated with magnetics. The nucleic acidmolecules were then eluted in hybridization buffer at 90° C. to denaturethe DNA component. Minimal loss was seen until the level of soil in thesample reached 32 milligrams per 100 micro liters (lane 8) where thesolution becomes viscous and particle movement is difficult under thecurrent test conditions. It is understood that by increasing thedisrupting power, modifying the solution, or changing the disruptingparticles size or characteristics results could be optimized forextremely contaminated samples.

Preparation of Magnetite Clusters

A first solution of ferric chloride (0.8M), ferrous chloride (0.4M) andhydrochloric acid (0.4M) was mixed and 0.2 micron filtered. A secondsolution was prepared with 72 ml of ammonium hydroxide (30%) with waterto make 1 liter.

1 ml of the ferric/ferrous chloride solution was added with stirring to20 ml of the ammonium hydroxide solution. Stirring was continued for 15seconds. The solution (in a 20 ml vial) was placed on a strong magnetand allowed to stand for 1 minute, after which all the product waspulled to the bottom of the vial. The clear supernatant liquid wasdecanted, replaced with water, mixed, and placed near the magnet. Againthe product was pulled to the bottom of the vial. This process wasrepeated three times to wash the product free from any residual ammoniumand iron salts. The vial was then filled with 20 ml of water andultra-sonicated for 5 minutes at 4 watts power. The suspension was thenfiltered through a 1 micron glass filter to give a stable suspension ofmagnetite nanoparticles that remain in suspension until pulled down bymagnetic forces or centrifugation.

Example A

Three fruit flies were placed in each of two 1.5 ml Eppendorf tubes. Onewas loaded with 100 microliters of a mixture of 100 mM TRIShydrochloride (pH 7.5), 1.5% dextran sulfate and 0.2% sodiumdodecylsulfate (SDS). The other was loaded with 100 microliters ofisopropyl alcohol and 10 microliters of 20% sodium dodecylsulfate. Bothtubes were loaded with 10 microliters of 0.6% magnetite nanoparticles inwater. Both tubes were sonicated at 20 kHz for 45 seconds (2 watts).Then 1 ml of isopropyl alcohol was added to the first tube and ½ ml ofisopropyl alcohol was added to the second tube. The magnetic pellet wascollected by a permanent magnet, the supernatant liquid decanted and 50μL of 100 mM sodium phosphate was added to each tube, the pelletresuspended by repetitive pipetting, then incubated at 95 degrees C. for2 minutes. The pellet was again collected on a magnet and the eluted DNAwas run on a 1% agarose gel at 77 volts in TEA buffer. A DNA ladder wasalso run on the gel.

As shown in FIG. 39, the gel was stained with ethidium bromide andphotographed with 302 nm excitation and a 610 nm filter over the camera.The purified DNA is clearly visible on the photograph. The top lanerepresents the second tube, the middle lane represents the first tubeand the bottom lane represents a DNA ladder.

Example B

Four tubes, each with three fruit flies, 100 microliters of buffer and10 μL of 0.6% magnetite nanoparticles were sonicated for 30 seconds at 5watts at 20 kHz. The DNA was collected, eluted, run on a gel, stainedand photographed as in Example A and shown in FIG. 40. The four bufferswere as follows:

1. 100 mM TRIS, 1.5% Dextran sulfate and 0.2% SDS

2. Isopropylalcohol (IPA)

3. 90% IPA, 1% dodecylbenzenesulfate, 9% water

4. 90% IPA, 1% polyacrylic acid sodium salt, 9% water

Example 13

Portions of yeast, grass and blueberries were sonicated in 100 mM TRIS,1.5% Dextran sulfate and 0.2% SDS as in Example A. The purification, geland photograph were as in Example A and are shown in FIG. 41.

Example C

Three 1.5 ml Eppendorf tubes each containing about 10 billion E. colicells and 33 mg of glass beads (100 micron diameter) and 40 microlitersof 0.5 molar sodium phosphate, pH 7.5 were sonicated for 15, 30 and 60seconds at 40 kHz, 10% amplitude with a 4 mm sonic tip inserted into thetube. The purification, gel and photograph were done as in Example A andare shown in FIG. 42.

This example shows that longer sonication times do not change the sizedistribution, i.e., that steady state conditions apply.

Example D

In this example, DNA is recovered from increasing volumes of a bacterialcell culture using two standard methods—the commercial QIAGEN kit forDNA recovery and the textbook Phenol/Chloroform method. These werecompared to the method given in Example A, using 0.2% SDS and 0.5 Msodium phosphate as the buffer. The results are shown graphically inFIG. 43.

The graph shows that the method of this invention is superior to boththe QIAGEN kit and the phenol/chloroform method.

Protective Buffer Example

In this example a comparison of protective buffers for DNA shearing byultrasonication are shown in FIG. 44.

5 μL G1 plasmid DNA solution containing 5 μg of DNA were mixed with 50μL buffer with 44 mg of zirconia beads of approximately 100 micron sizein a 1.5 ml eppendorf tube. The tube was inserted into the socket of aBranson SLPt 40 kHz ultrasonicator. The sonicator was run at 50%amplitude for 12 minutes with a pulsed cycle of 10″ on and 20″ off.After sonication, a 20 μL portion of the mixture was eletrophorized on a1% agarose gel at 100 volts in TAE buffer. All buffers were adjusted toa pH between 7 and 8. A DNA ladder was run on both sides of the samplelanes. The lanes contained:

Lane 1. TE (Tris-(hydroxymethyl)aminomethane) with EDTA (ethylenediamine tetra-acetic acid)

Lane 2. 10 mM Tris-(hydroxymethyl)aminomethane

Lane 3. 500 mM sodium phosphate

Lane 4. 50 mM sodium phosphate

Lane 5. 60 mM sodium citrate

Lane 6. 3% sodium chloride

This example shows that high ionic strength buffers, such as metal saltsare effective in protecting the DNA during sonication. The buffer allowsfor larger DNA fragments in a steady state sonication. Lower ionicstrength buffers such as Tris-hydroxymethyl aminomethane are lessprotective and yield smaller DNA fragments suitable for particularapplications.

In one embodiment, the size stabilizer is a protective high ionicstrength buffer including soluble salts from cations including the Group1 and Group 2 metals of the periodic table with anions from Group 7 ofthe periodic table as well as more complex anions exemplified bysulfates, phosphates, and acetates. In another embodiment the buffer iscapable of being stable and soluble at pH values between 7 and 8. Thesoluble concentration of the buffers is, in one embodiment, greater than1%. In another embodiment, the concentration is greater than 5%.

Surfactant Examples

Two fruit flies were placed in each of 3 eppendorf tubes containing 25of 100 micron glass beads from Biospec Products. To the first tube, 100microliters of water was added. To the second tube, 100 microliters of1% sodium dodecylsulfate was added. To the third tube, 100 microlitersof 1% sodium dodecylbenzenesulfate was added. All three tubes weresonicated for 2 minutes on power level 2 on a Branson Sonifier 150,placing the tube into the threaded orifice of the ultrasonic converterwhere the tips are normally threaded into the converter. The power metershowed an initial reading of about 8 watts which dropped during the 30seconds to about 4 watts, which level continued during the remainder ofthe sonication time. After sonication, 20 microliters of the fluid abovethe glass beads was removed and placed in the wells of an agaroseelectrophoresis gel, made with TAE buffer. A DNA ladder was included inthe first lane to determine the size of the sonicated DNA fragments.After electrophoresis at 70 volts for 90 minutes, the gel was soakedwith gentle agitation with an ethidium bromide solution. Then a blacklight photograph of the gel was taken, as shown in FIG. 38.

Referring to FIG. 45, the first lane above the DNA ladder shows thewater sonication result. A low yield of DNA is seen, and the fragmentsare smaller than 400 base pairs. The second lane above the ladder showsthe sodium dodecyl sulfate sonication result. The yield of DNA is muchhigher, and the fragment sizes range from 300 to 2000 base pairs insize. The third lane above the ladder shows the sodiumdodecylbenzenesulfate result. Again, the yield of DNA is high, evidencedby the bright spot on the photograph, and the size range is from 300 to1000 base pairs. This example shows that sonication in the presence of aselected surfactant can provide a high yield of DNA in a limited sizerange from a live source such as fruit flies.

Exemplary Process

A sample in the form of a liquid or solid is loaded into the centerreservoir using one of several specialized covers designed for aspecific sample type or source. The cover may contain, for example, alance for blood collection, a frit to exclude large debris, or a set offilters to pass only certain materials, cells or pathogens of within adesired size range into the cartridge central chamber.

The instrument run operation begins by extracting a sufficient amount oflysis buffer from its storage reservoir and pumping it into the centersample reservoir. Mixing with the loaded sample is performed byalternating the pumping direction of a volume slightly less than that ofthe combined liquid volume of the sample and lysis buffer.

For certain types of samples which are difficult to chemically lyse, anultrasonic horn integrated within the instrument drive unit is activatedto drive glass bead beating of the lysis mixture. With or withoututilizing the glass-bead beating step, the lysis mixture is incubated atambient temperature for 5-10 minutes to allow chemical lysis of thesample. While incubating the sample, certain preparative actions fordownstream processing steps can be performed. For example, 100 μl of DIwater is added to reconstitute dried P30 size-exclusion resin packagedinside the desalting pod.

Upon completion of the chemical lysis incubation, the lysate mixture isextracted through a 30 μm filter and loaded into a reservoir containing15 μl ferrite magnetic particles (prepared as a suspension in a viscousliquid such as polyethylene glycol (PEG), sugars, glycerol, or otherorganic polymers such as PVP. The solutions are mixed and allowed toincubate for 5 minutes. During this incubation, the nucleic acids(DNA/RNA) are bound to the magnetic particles.

The sample-magnetic particle complex is extracted and passed through achannel that passes over a magnet in small volume pulses (1 to 10 μl).Each pulse is allowed to dwell for at least 2 seconds over the magnet.This ensures sufficient time for the magnetic particles to be attractedto the magnet. The residual buffer is flushed through the channel andinto the cartridge waste reservoir.

The ferrite reservoir chamber is flushed with an appropriate volume ofwater (40 μl to 200 μl) that is then passed over the magnet to collectany residual nucleic acid-ferrite complex. This step also rinsespreviously bound magnetic particles of contaminates and any remainingbuffer. The magnetic channel is purged with air to remove any residualrinse water.

In order to release bound nucleic acids efficiently from the ferrite, aheater is activated and allowed to stabilize at 80° C. A 22 μl volume ofelution buffer (50 mM sodium phosphate with 1 to 15% organic polymer (2%PVP preferred) is withdrawn from its reservoir and then pumped into themagnetic channel to flow over the ferrite/sample pellet. The cartridgevalve is rotated a few times around away and back to the magnet toloosen the ferrite/sample pellet. The ferrite/sample pellet is rotatedover the heater and allowed to incubate at 80° C. for 5 minutes. Duringthis time, the sample is released from the magnetic particles into theelution buffer.

The magnetic channel is then rotated back to position it over the magnetwhere the magnetic particles are once again attracted to the magnet.This step allows the released NA material to be separated from thedepleted ferrite particles by extracting the eluate solution from thechannel. Removal of the phosphate salts any other contaminants isachieved by pumping the eluate into the desalting pod. Because of thevolume of the eluate is small, a minimal amount of DI water (10-100 μl,55 μl utilized initially) is used to push the sample through the pod.The purified sample exits the pod through a 0.2 μm filter and spillsinto an overflow reservoir. The sample is now ready for PCR (or otherprocessing that may include: restriction digests, phosphorylation,de-phosphorylation, ligation, nuclease treatment).

Nucleic Amplification

The purified material derived from the cartridge sample-prep processingis extracted out of the desalting overflow reservoir, taking a volume of45 to 90 μl. This solution is used to set up either one or two separateamplification reactions. For example, to perform two independent PCRreactions, the purified nucleic acid material is divided by pumping halfthe solution into the PCR Mix 1 reservoir to reconstitute a lyophilizedPCR pellet having one particular set of amplification primers; theremaining half of the solution is used to reconstitute the lyophilizedPCR pellet in the PCR Mix 2 reservoir that has a different set ofamplification primers.

Following PCR pellet reconstitution, 39 μl of each PCR Mix is dispensedinto its designated PCR channel (mix 1 into channel 1, and mix 2 intochannel 2), as viewed on the underside of the cartridge rotor valve. Theinstrument pre-warms and stabilizes the two heaters to a starttemperature (for example, 45-65° C. for RT-PCR and 93-98° C. for PCR).Subsequently, the cartridge valve is rotated so the PCR channels arealigned over the heaters, while the inlet and outlet ports are blockedto contain pressure that arises from the heating of the PCR solution.Standard methods and variations for both Reverse Transcription-PCR(RT-PCR) and PCR reactions (i.e. two or three step PCR) can now beperformed according to the process best suited for optimal productgeneration, and which avoids artifact products or that is leastsensitive to problematic inhibitors that may be carried over fromsample-prep. When PCR cycling is completed, the instrument turns off theheaters to allow the temperature in the channels to cool to ambient. Themachine rotates the valve and uses the syringe pump to extract 30 μl ofPCR sample 1 and 2 from their respective channels. These volumes arecombined into a PCR Mix reservoir for storage and preparation fordetection.

Detection Method Steps

A portion of the pooled PCR products (10-50 μl) is taken from the totalvolume held within the PCR Mix 2 reservoir and then dispensed into amixing chamber. Next, hybridization buffer (250 mM NaP_(i)/0.1% SDS) isaspirated from its storage reservoir and mixed with the selected amountof PCR solution in the mixing compartment. For optimal performance, thesurface of the sensor microchip must be pre-wet using a rinse of thehybridization buffer only and then be allowed to reach and maintain 60°C. for a few minutes before pumping in 70 μl of the hybridization testsolution that contains the pooled PCR reaction output material.

Once dispensed into the reaction chamber, target derived PCR productshybridize to appropriate sensor electrode regions on the surface of thetest microchip. Multiplexing capability is achieved due to the sequencespecificity imparted by utilizing different capture oligonucleotideprobes, which have been spatially addressed as an array overlaying thepatterned groups of independent sensors on the microchip. The durationof hybridization reaction can be from 30 to 600 seconds, depending onpreference for greater sensitivity or a shorter time to result. The testhybridization solution can be held static on the surface for theduration of the hybridization, or be flowed in a pulsed or continuousmanner. Afterwards, to remove any remnants of the amplification reaction(non-hybridized nucleic acids, PCR products, or primer oligonucleotides,and dNTPs and any reaction by-products) the reaction chamber is rinsedwith one 130 μl aliquot of hybridization buffer only solution, with thechip heater maintained at 60° C.

Incubation with catalyst solution (noble metal ionic compound, colloidor cluster) follows the rinse immediately. Colloid or cluster catalystsmay be functionalized with oligonucleotides, antibodies, or other targetgeneric or specific recognition molecules. The catalyst reagent may beheld static, or flowed in a pulsed or continuous manner over thereaction surface for 30 to 600 s at 25-65° C. The catalyst is rinsed offwith 2×100 μl aliquots of hybridization buffer. The chip surfacetemperature is then increased to 68° C. to prepare for development.

A lyophilized pellet of developer chemicals is hydrated in 96 μl of DIwater and 10 to 60 s of mixing within the developer reservoir chamber.The reconstituted developer is aspirated and then dispensed at a veryslow flow rate for 105 s. About 25 μl of developer will react with thechip surface over this time. The chip temperature is lowered to 50° C.and rinsed with 100 μl of H₂O. The syringe aspirates air from adesignated chamber, and dispenses it over the chip surface to dry it. A60 s delay with the heater at 50° C. ensures the surface is dry. Theresistance of the sensors is measured.

Exemplary Cartridge

The cartridge comprises two plastic pieces: a main body and an internalrotor (see FIG. 46). The cartridge body has a syringe plunger actuatedby a screw type motor to move fluids. A reaction chamber is located onthe opposite side of the cartridge body with one wall of the cartridgeformed by the microchip sensor. Multiple reagent chambers are built intothe top of the cartridge rotor and reaction chambers and flow channelsare molded into the bottom of the cartridge rotor. The chambers aresealed with plastic seals on the top and bottom of the rotor.

The cartridge sits on a drive mechanism that rotates the inner plasticcomponent to align various rotary valve channel ports with ports in theouter housing for the syringe and detection chamber. A second motor inthe assembly is used to drive the cartridge syringe plunger to movereagents and solutions. The drive assembly is depicted in FIG. 47A andshown with a cutaway view. For sample and organism/cell disruption, anultrasonic horn is integrated within the drive assembly and contacts theunderside of the cartridge. The drive platform features an embeddedmagnet and two resistive elements. The magnet serves to concentratebiological molecules associated with paramagnetic particles, while thepair of regulated heaters 490 and a chill plate 492 (see FIG. 49) isused to heat/cool and control temperatures of specific channel areas onthe bottom of cartridge for biological reactions to be performed. Thechill plate is a thermally conductive material, such as a metal plate,that transfers heat away from the disposable insert.

Filtering

To provide flexibility with respect to various types of input materialsuch as blood versus insect samples, the cartridge is designed withfiltration pods (FIG. 47C) that can be varied to match the sample needswithout changing the basic design. Modular pods are used to incorporatefiltration and column separation into the cartridge. Filters areattached to rings and columns are prefabricated in cylinders that arethen welded into the cartridge. The number and type of pods can beselected based upon the target sample. For example samples can bepre-filtered to remove large debris particles prior to disruption,whereas insect samples would not be filtered prior to disruption.

Desalting:

An insert pod can be placed into the rotor valve (see FIG. 47C) thatcontains a desalting resin/matrix material. Flowing partially purifiedsample through the insert will remove any ions that could interfere withbiological reactions (i.e. iron inhibition of PCR amplification).

The insert must also manage/control the flow of fluid during thedesalting process. Surface properties of the plastics, reservoirgeometries, and air bubbles can affect the flow of the fluid. Alternatedesign concepts have been made to minimize these aberrations (FIGS.48A-D). The left design (FIGS. 48A and 48B) utilizes a cap which directsthe fluid over the side of the pod. Without the cap, the hydrophobicnature of the plastic can cause variability of the volume required tocause spill over. Similarly, the right design (FIG. 48C and FIG. 48D)utilizes a smaller exit port, which requires less volume to breaksurface tension.

Viral Separation

The sample preparation system should provide an ability to enrichviruses and bacteria from complex samples to improve the sensitivity ofdetection systems and the efficiency of gene sequencing. Automatedgenomic sequencing is becoming more cost effective and provides the bestcapability for identification of unknown pathogens. The latestgeneration of gene sequencers, such as Illumina's MySeq, provide thecapability to sequence viruses and bacteria in a matter of hours.However, sample preparation is critical for efficient genes sequencing.In particular, it is necessary to enrich a sample for the targetpathogens by isolating the viral and bacterial material away fromeukaryotic material found in samples, such as human blood samples orinsects. Otherwise the much larger eukaryotic genomes will dominate thesample and will necessitate sequencing much larger volumes of materialto identify the viruses or bacteria. Since a key advantage of sequencingis the ability to identify previously unknown pathogens, it is importantthat the enrichment process does not rely on prior knowledge of thepathogens.

The disclosed approach may be selectively enrich the purification oftargeted pathogens in a sample's background of genetic materials (suchas host cells). The exemplary cartridge features a special chamberdesigned to accommodate a column insert (see FIG. 46). Originally, adesalting column was intended to be positioned into this compartment.

After disrupting the sample mechanically, the material is passed througha first filter to remove intact eukaryotic cells. For bacteria, thefilter will have 2 to 4 micron pores, allowing the bacteria to pass butcollecting any eukaryotic cells. For viruses, the filter can have poresas small as 200 nm. The bacteria or viruses are then separated andconcentrated using a filter with smaller pores to capture the pathogens.A filter with 200 nm or less openings will be used for bacteria and afilter with 30 nm or less openings will be used to capture viruses or acombination of bacteria and viruses. In this step, the bacteria orviruses remain intact and nucleic acids released from rupturedeukaryotic cells will be washed through the filter. The washed virusesand/or viruses will then be lysed and nucleic acids can then movethrough the filter to be processed further.

The primary fraction of the sample will pass through a filter to removelarge debris and whole eukaryotic cells, and then the bacteria orviruses will be captured using a novel filter based on a pnc-Si or tracketched membrane which will capture particles greater than 30 nm in size.These membranes allow for fine control of the size of material allowedto flow through and minimizes loss due to material being trapped in thefilter.

Porous nanocrystalline silicon (pnc-Si) membranes represent arevolutionary advance in membrane technology. The most significantstructural characteristic of pnc-Si is its molecular scale thickness(10-50 nm), which results in transport resistances and losses that areorders-of-magnitude lower than conventional membranes that are 100-10000times thicker than pnc-Si. Because transmembrane resistance to bothconvective and diffusive transport increases is proportional to membranethickness, molecularly thin membranes effectively minimize a criticalparameter that adversely affects membrane permeability. Consequently,the permeability of pnc-Si to water, gas and diffusing species are thehighest reported for experimental or commercial nanoporous membrane. Inmany practical settings, pnc-Si membranes offer transport resistancesthat are so small compared to other components in the system, that theycan be neglected. Despite the nanoscale thickness of pnc-Si, themembranes are mechanically robust and can be manufactured in largequantities.

The resolution of separations is also known to improve for thinnermembranes, and pnc-Si membranes have been shown to separatenanoparticles and proteins with resolutions exceeding 5 nm regardless ofthe mode of transport. The membranes are also modifiable through silanechemistries that can be used to graft polymers to reduce protein bindingand fouling, or manipulate surface charges for charge-based separations.Additionally, ultrathin membranes minimize sample loss throughabsorption to internal surfaces, providing a low loss membrane forprocesses involving low concentrations and small volumes.

Selective enrichment and cleaning of these pathogens should improvedownstream assay performance by virtue of a more effective removal ofinhibitors and limiting the presence of extraneous eukaryotic nucleicacids prior to a subsequent lysis step. Nucleic acid binding and elutionsteps with our magnetic particles perform better when clean nucleicacids has been the input.

Loaded materials in the collection column will be washed with rinsebuffer, and then undergo our sample processing. The excluded, retainedand passed-through column materials will be titrated into amplificationreactions to determine whether certain pairings of exclusion membranesbest enrich virus over arthropod material. As virus becomes enriched inthe column-retained fraction, RT-PCR detection should persist or improvewith the most dilute manipulations of a titrated series; detection ofarthropod DNA should diminish with effective fractionation by a filterset.

Sample Input Different cartridge lid accessories have been designed toaddress varying types of sample input. For example, FIG. 50 shows acartridge cover designed for input of insect vectors or tissue samples,where the sample material may need to be crushed prior to sonication.Sample Input—Liquid (e.g. Blood) Collection:

The device shown in FIG. 51 utilizes a plastic bulb embedded with alancet or other sharp puncturing device. When pressed, the bulb beginsto deform creating pressure. A check valve on the bulb opens relievingthe pressure as the bulb continues to collapse. Once the bulb assufficiently deformed the embedded lancet pierces the thin film seal andsubsequently the subject's skin/membrane. Because of the check valve,there is very little to no air in the bulb. This prevents an air pocketfrom forming just below the subject's skin/membrane allowing maximumfluid extraction. The bulb is then released allowing its naturaltendency to return to its original shape. Doing so, the vacuum createddraws in fluid from the puncture wound. The extracted volume iscomparable to the displaced bulb volume (minor sealing leaks).

Dispensing the collected sample is done by simply affixing the bulb unitto the base where the check valve can be blocked. Pressing the bulb,again, creates pressure but because the check valve is blocked thesample is forced back through the original puncture hole and into acollection vessel.

The device comprises three parts; the base, the vacuum mechanism, andthe plunger. The base comprises of a soft plastic ring/perimeter thatacts like a gasket when in contact with a surface to insure good vacuum.One edge of the base will have a tab that is used to plug a check valvelocated in the vacuum mechanism. This tab prevents the check valve fromopening when the device is in the load or closed position. The base mayhave a hinge that connects it to the vacuum mechanism or an adjacentdevice. The base may have a depressed region that acts to hold/containthe sample. Lastly, the floor of the base is a thin plastic film thatcan be easily punctured.

The vacuum mechanism comprises a soft plastic bulb that can return toits original shape after being compressed. An integrated check valve isformed at the edge of the bulb in order to prevent air pocket formation.Embedded at the apex of the bulb is a lancet that is used to puncturethe subject. A thin plastic film (similar to the base) separates thebase from the vacuum bulb. Above the bulb is a planar piece of plasticthat is used to evenly compress the bulb. This piece is attached to theplunger. If required, a spring can be added to aid in the reformation ofthe bulb.

The plunger is simply a leverage tool to aid in operation. It can bemodified to twist and lock in the depressed state to ensure completecompression.

A modification can be made to the device to prepare difficult mediumsfor analysis by replacing the lancet with a crushing or chopping edge.In this scenario, a sample such as an insect or piece of tissue is firstplaced in the recessed pocket in the base of the device. The vacuummechanism is then attached to the base where the cutting/chopping bladesare above the sample. Since a vacuum is not required for this operation,the seal in the vacuum mechanism is not required. The bulb is thenpressed, lowering the blades onto the sample. Releasing the bulb allowsthe blades to rise for repeated cuts. When sufficient processing hasbeen completed, the blades and sample can be pressed with enough forceto break the bottom seal pushing the sample into another vessel.

Multiple bulbed configurations connected by a common channel can allowfor grouped collection and analysis (FIG. 51). To do this, an additionalcheck valve must be included between the bulb and the channel. Thisprevents air from being actuated into the channel when the bulb ispressed. To do this, an additional flap can be molded along with thebulb. This flab collapses into the channel when it is depressedpreventing flow. The flap mechanism can easily be linked with themechanism used to compress the bulb.

To combine and dispense the samples to another vessel, the apparatus isfitted into a tool similar to what is used to collect the sample(hammer/plunger compressor). The difference is that all bulbs arecompressed while keeping the common channel flap valves open. The commonchannel leads to an output port that is mated to the collection vessel.This port is plugged until the fluid is dispensed. With the port opened,the collection vessel attached, all the bulbs are compressed at once.Because the channel flaps are left open, the fluid is pushed through thecommon channel and into the collection vessel. The pinholes under thecollection bulbs are sealed by the tool's base when the hammer/plungeris compressed preventing leaks.

Multi-Sample Collection Tool—Sample Collection

As shown in FIG. 51 and FIG. 52, a multi-sample collection disk isinserted into the load side of the tool (button side) with the breakawaytab facing up. The disk is oriented so a collection bulb is beneath thehammer tab. The user inserts the sample (e.g. an animal ear) between thedisk and the bottom plate and squeezes the bottom lever. This raises thebottom plate pinching the sample between it and the disk. Additionally,the compressed lever causes the channel flap valve hammer to pivot andlower onto the channel flap valve and consequently closing it. Whilestill squeezing the lever, the top lancet button is pressed, compressingthe bulb and piercing the sample with the embedded lancet. The button isreleased (spring assisted) and the bulb returns to its original formcreating a vacuum and drawing in the sample. The lever is release, thechannel flap valve hammer is raised, and the bottom plate is releasedfrom the sample. The disk is then rotated to the next available bulb forfurther collection.

Multi-Sample Collection Tool—Sample Transfer

The full, multi-sample collection disk is inserted into the transferside of the tool with the breakaway tab facing down. The disk is alignedso that all the bulbs are under a compression hammer. The breakaway tabis removed creating an open port. An appropriate vessel is attached tothe bottom plate (twist and lock). The lever is squeezed and the lowerplate/vessel is raised and compressed against the disk bottom (thedispense port has a piercing edge allowing thin film seals to bebroken). As the lower plate is forced against the disk, the hammerscompress the bulbs and the duck/flap valves forcing the samples throughthe common channel and into the vessel. The lever is released and thedisk is removed.

First Exemplary Sample Prep Lid (Solid)

Referring to FIG. 53, this method uses traditional capillary action towet an absorbing solid with the sample (blood) before being transferredto a subsequent vessel. Like many blood analyzers, this apparatuscontains a lancet for puncturing the skin which allows the sample to beabsorbed into the material. The material can be anything that absorbsand retains a liquid sample (paper, cellulose matrix, etc). The materialcan be cut, pleated or woven in any manner to adjust the collectedvolume.

The device comprises four parts; the body, the sample ring, the lancetand the sample ring ejector. The body contains and braces the threecomponents inside providing structural integrity. The sample ring is asmall frame that the absorbent material is stretch around holding it inplace. The sample ring is press fitted into the body but is intended tobe removed with sufficient force. The lancet is spring loaded andconnected to a handle/button. The lancet also has a unique key typepiece that allows it to toggle between actuating the lancet and ejectingthe sample ring. The sample ring ejector is a cylinder shaped piece thatsits directly upon the sample ring. The lancet and its key piece, passthrough the center. Under static and lancing conditions, the key pieceslides inside the lock housing in the sample ring ejector. However, ifthe user pulls up and rotates the handle 90°, the key piece is removedfrom the lock housing and rests on top of the sample ring ejector. Theuser can then press down on the handle, forcing the sample ring ejectoronto the sample ring, which causes the sample ring to dislodge. Tomaintain a sterile environment, each end of the housing can be sealedusing a traditional heat film. The film is removed prior to lancing thesubject.

An alternate design can be employed that does not use a key and lockmethod. Rather, the lancet spring is placed between the handle, nowbutton, and the sample ring ejection piece (instead of the body). Thespring still actuates when the button is pressed but is no longer usedto eject the sample ring. To eject the sample ring, the sample ringejector piece is connected to a separate button/lever through ports onthe button plate.

This method can be modified to be used in multi sample collection disks.The tool would comprise of a sample clamp and two buttons to actuate thelancet and sample ring ejector. Additionally, an auto indexer and avessel clamp can be added.

Explanation of Operation

For the first device, the user removes the protective film from thefilter ring and places the sample ring side of the device onto thesubject. The user then presses down on the handle to actuate the lancetand puncture the subject. As the sample leaks from the wound, the samplering absorbs a fixed amount of liquid. The device is then placed over acollection vessel. The user pulls and rotates the handle 90° and thenpresses down forcefully causing the sample ring to eject into thevessel. The method is the same for the alternate device except for thesample ring ejection, which requires just a press of a button.

Second Exemplary Sample Prep Lid (Solid)

The cover of FIG. 54 is very similar to the previous design with theexception that the filter ring is not ejected into the sample prepchamber. Instead, the lid is reaffixed to the cartridge wherein thefilter matrix is submerged into the chamber where it can be extracted.To maintain a sterile environment, the lid is stored separately and aplastic sleep is placed over the syringe/filter region (syringe cap).

Third Exemplary Sample Prep Lid (Solid)

As shown in FIGS. 55A, 55B and 55C, this cover is very similar to thesecond exemplary sample preparation lid in terms of mechanicaloperation. The sample is still collected by the prick, absorb, and loadmethod as outlined above. The first difference is the ergonomic design.The collection tool has been contoured to a more “syringe-like” formfactor to simplify handling and use (FIGS. 49A, 49B and 49C). The secondmodification comes in the way of the mechanical actuation of the lancet.To ensure single use only, the actuation mechanism has been designed toactuate once. A cover with a build-in lance is shown in FIG. 56 thatdoes not use a syringe-like form factor.

Multi Sample Collection (solid) v.1B

As mentioned earlier, the solid sample prep v.1B apparatus can bemodified to collect multiple samples. This is done by incorporatingmultiple syringe/ejector/sample ring modules into a single, disposabledisk. The disk can be loaded into a collection tool similar to thedesign in FIG. 53. Like the tool in FIG. 53, the tool has a pair ofclamps for stabilizing the sample. The lancet and the ring ejector areactuated via a button. The tool is also capable of indexing the disk tothe next free position after collecting a sample. Tocollect/combine/remove the samples, a vessel is attached to the tool. Asthe tool indexes through available positions, the used regions rotateand eventually pass over the collection vessel where they can be ejectedinto the vessel. Once all the free regions have been used, the usercontinues to index/eject the disk until all the collected samples areloaded into the vessel. Once this occurs, the disk can be removed andstored/discarded. To ensure all the samples are collected, the user isunable to remove the disk until it indexes to the “complete/unload”position. For simplicity, the lancet, the ring ejector, and the indexfunctions can be combined.

Sample Prep Lid (Liquid Concentrator)

In order to handle liquid samples that are larger than the 100 μL, amodification to the absorbent method can be used. Assuming the desiredsample has been collected in a vacuum tube (i.e. blood) or some othervessel; the sample can be extracted and passed through a series offilters (10 μm and 0.2 μm). The final (smallest) filter acts a trap andcontains the desired nucleic acids, spores, etc. And like the absorbentmethod, the trap filter can be ejected into an ex

While the invention has been described with reference to particularembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from thescope of the invention.

Therefore, it is intended that the invention not be limited to theparticular embodiments disclosed as the best mode contemplated forcarrying out this invention, but that the invention will include allembodiments falling within the scope and spirit of the appended claims.

What is claimed is:
 1. A method for preparing a nucleic acid sample using a disposable cartridge, the method comprising the steps of: inserting a biological sample into a disrupting chamber of a cylindrical insert that is rotatably connected to a cartridge body, the disrupting chamber being fluidly connected to a first port in an edge of the cylindrical insert, the cartridge body having a syringe mold connected to a plunger; activating a disruptor that supplies sonic energy to the disrupting chamber; permitting the sonic energy to disrupt the biological sample and release nucleic acid molecules while exposing the nucleic acid molecules to a size stabilizer to produce a nucleic acid solution; permitting the nucleic acid molecules to bind to magnetic nanoparticles; rotating the cylindrical insert to align the plunger with the first port and activating the plunger to remove the nucleic acid solution from the disrupting chamber; rotating the cylindrical insert to align the plunger with a second port which is fluidly connected to a wash chamber that is proximate a magnet; activating the plunger to inject the nucleic acid solution into the wash chamber; permitting the magnet to concentrate the magnetic nanoparticles with the nucleic acid molecules at a first location of the wash chamber; activating the plunger to remove a portion of the nucleic acid solution while leaving the magnetic nanoparticles with the nucleic acid molecules at the first location; removing the nucleic acid molecules from the magnetic nanoparticles to provide prepared nucleic acid molecules. 